EP2465936A1 - Synthèse enzymatique de statines et intermédiaires associés - Google Patents

Synthèse enzymatique de statines et intermédiaires associés Download PDF

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EP2465936A1
EP2465936A1 EP10015774A EP10015774A EP2465936A1 EP 2465936 A1 EP2465936 A1 EP 2465936A1 EP 10015774 A EP10015774 A EP 10015774A EP 10015774 A EP10015774 A EP 10015774A EP 2465936 A1 EP2465936 A1 EP 2465936A1
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pqq
enzyme
compound
dehydrogenase
dehydrogenation
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Lek Pharmaceuticals dd
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Priority to EP10015774A priority Critical patent/EP2465936A1/fr
Priority to CA2822331A priority patent/CA2822331A1/fr
Priority to JP2013545306A priority patent/JP6194251B2/ja
Priority to CN201180067928.6A priority patent/CN103403174B/zh
Priority to AU2011355209A priority patent/AU2011355209B2/en
Priority to US13/995,420 priority patent/US20130337485A1/en
Priority to EP11811536.9A priority patent/EP2655650B1/fr
Priority to PCT/EP2011/073412 priority patent/WO2012095244A2/fr
Publication of EP2465936A1 publication Critical patent/EP2465936A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings

Definitions

  • the present invention relates in general to the field of chemical technology and in particular to a process for preparing an active pharmaceutical ingredient (API) or intermediates thereof by using an enzyme.
  • the present invention relates to a preparation of HMG-CoA reductase inhibitors, known also as statins, wherein a certain enzyme is provided for catalyzing a particular step in the synthesis.
  • this invention relates to a process for preparing an intermediate by providing said enzyme.
  • the invention further relates to an expression system effectively translating said enzyme.
  • the invention relates to a specific use of such enzyme for preparing API or intermediate thereof, and in particular for preparing statin or interemediate thereof.
  • Synthetic routes are routinely performed by carrying out chemical reactions in vitro.
  • the chemistry can become complex and can require expensive reagents, multiple long steps, possibly with low yields because of low stereoseledivity.
  • a microorganism or its parts like for example enzymes, optionally together with synthetic process steps, it may be possible to put together complete synthesis pathways for preparing an API or an intermediate thereof, however, it is a challenging task.
  • HMG CoA reductase 3-hydroxy-3-methylglutaryl-coenzyme A reductase
  • a natural lovastatin is known, which has been superseded by semisynthetic pravastatin, simvastatin and later by completely synthetic atorvastatin, cerivastatin, rosuvastatin, fluvastatin, pitavastatin, bervastatin and dalvastatin.
  • statins characterised by INN name ending -statin.
  • statins share a characteristic side chain consisting of respectively a heptenoic or heptanoic acid moiety (free acid, salt or lactone) connected to a statin backbone (Scheme 1). Biological activity of statins is linked to this structure and its stereochemistry. Normally, multiple chemical steps are required to prepare the heptenoic or heptanoic acid moiety. The construction of this side chain still represents a challenge for a chemist.
  • Specific aspects of the present invention can be applied for preparing statins or their intermediates.
  • the present invention provides a process according to claim 1. Preferred embodiments are set forth in the subclaims.
  • the present invention further prodives a reaction system according to claim 9, a process for preparing a pharmaceutical composition according to claim 13, expression systems according to claims 14 and 15 and uses according to claims 18 to 21.
  • the oxidation or dehydrogenation of the compound of formula (II) performed with the enzyme catalyst as defined above according to the present invention is extremely favourable over the prior art chemical oxidation with Br 2 /BaCO 3 or the chemical catalytic dehydrogenation (e.g. Pt/C, Pd/C).
  • Chemical oxidants are not specific and thus need previous purification of lactol, otherwise too much reagent is consumed in oxidation of the side products or reagents, or even solvents.
  • the purification of lactol is demanding and requires substantial amounts of a solvent for extraction, which is linked to the fact that lactol is normally hydrophilic and is hardly extracted to the organic solvent, such as for example ethylacetate.
  • lactols such as compound (II) are non-natural type compounds, they have surprisingly been found to work as effective substrates for the enzymes particularly chosen and disclosed herein. Based on this finding, use of the enzyme capable of catalyzing oxidation or dehydrogenation provides a valuable tool for generally preparing a synthetic API or synthetic intermediate thereof.
  • the substrate within the use of the present invention for preparing a non-natural, synthetic API or intermediate thereof will therefore be different from naturally occurring ones of the enzyme capable of catalyzing oxidation or dehydrogenation, thereby excluding for example natural substrates selected from ethanol, methanol, acetaldehyde, acetic acid, naturally occurring sugars or amino acids, or sugar acids derived from sugars, including monosaccharides having a carboxylic group such as gluconic acid.
  • mono- or bicyclic aryl group refers to any mono- or bicyclic, 5-, 6- or 7-membered aromatic or heteroaromatic ring, such as for example pyrolyl, furanyl, tiophenyl, phenyl, imidazolyl, pyridinyl, piridazinyl, indolyl, kinolinyl ftaliminyl and benzimidazolyl.
  • aryl as used herein, if not stated otherwise with respect to particular embodiments, includes reference to an aromatic ring system comprising 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring carbon atoms.
  • Aryl can be phenyl but may also be a polycyclic ring system, having two or more rings, at least one of which is aromatic. This term includes phenyl, naphthyl, fluorenyl, azulenyl, indenyl, anthryl and the like.
  • mono- or bicyclic heterocyclic group refers to any mono- or bicyclic, 5-, 6- or 7-membered saturated or unsaturated ring, wherein at least one carbon in the ring is replaced by an atom selected from the group of oxygen, nitrogen and sulphur.
  • mono- or bicyclic heterocyclic group are oksazolyl, tiazolyl, isotiazolyl, morfolinyl.
  • heterocycle includes, if not stated otherwise with respect to particular embodiments, a saturated (e.g. heterocycloalkyl) or unsaturated (e.g. heteroaryl) heterocyclic ring moiety having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, at least one of which is selected from nitrogen and oxygen.
  • heterocyclyl includes a 3- to 10-membered ring or ring system and more particularly a 5- or 6-or 7-membered ring, which may be saturated or unsaturated; examples thereof include oxiranyl, azirinyl, 1,2-oxathiolanyl, imidazolyl, thienyl, furyl, tetrahydrofuryl, pyranyl, thiopyranyl, thianthrenyl, isobenzofuranyl, benzofuranyl, chromenyl, 2H-pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyr
  • a saturated heterocyclic moiety may have 3, 4, 5, 6 or 7 ring carbon atoms and 1, 2, 3, 4 or 5 ring heteroatoms selected from nitrogen and oxygen.
  • the group may be a polycyclic ring system but more often is monocyclic, for example including azetidinyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, oxiranyl, pyrazolidinyl, imidazolyl, indolizidinyl, piperazinyl, thiazolidinyl, morpholinyl, thiomorpholinyl, quinolinidinyl and the like.
  • heteroaryl may include an aromatic heterocyclic ring system having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, at least one of which is selected from nitrogen and oxygen.
  • the group may be a polycyclic ring system, having two or more rings, at least one of which is aromatic, but is more often monocyclic.
  • This term includes pyrimidinyl, furanyl, benzo[b]thiophenyl, thiophenyl, pyrrolyl, imidazolyl, pyrrolidinyl, pyridinyl, benzo[b]furanyl, pyrazinyl, purinyl, indolyl, benzimidazolyl, quinolinyl, phenothiazinyl, triazinyl, phthalazinyl, 2H-chromenyl, oxazolyl, isoxazolyl, thiazolyl, isoindolyl, indazolyl, purinyl, isoquinolinyl, quinazolinyl, pteridinyl and the like.
  • mono- or bicyclic alicyclic group refers to any mono- or bicyclic, 5-, 6- or 7-membered alicyclic ring.
  • the term "salifyed” or "a pharmaceutically acceptable salts" in a context of the compound of formula (I) or (II), API or statin, which can be optionally substituted, as used herein refers to the compound or statin in a form of a saft, such as potassium, sodium, calcium, magnesium, hydrochloride, hydrobromide, or the like, that is also substantially physiologically tolerated.
  • the compound of formula (I) or (II), API or statin can be salifyed or brought in the form of a salt by mixing the compound of formula (I) or (II), API or statin or intermediate thereof, with an acid or a base, optionally in an aqueous or organic solvent, or a mixture thereof. Preferably the solvent is afterwards removed.
  • esterifying or “esters” in a context of the compound of formula (I) or (II), API or statin, as used herein refers to the compound of formula (I) or (II), or statin, with at least one ester bond in their structure.
  • ester bond or esterifying the compound can be achieved by coupling the compound of formula (I) or (II), API or statin or intermediate thereof, in the event the compound or statin contains hydroxyl group, with an carboxylic acid or a phosphate group containing compound.
  • API or statin or intermediate thereof contain carboxylic or phosphate group, it can be achieved by coupling it with a hydroxylic group of another compound.
  • stereoselectively resolved is used herein to refer to any method known to the skilled person in the field of separating a mixture of stereoisomers, preparatory chemistry of stereospecific compounds, or analytics.
  • the stereoisomers can be obtained for example by HPLC, wherein stereoselective column is used.
  • Stereoselective columns are known in the art.
  • an enzyme capable of catalyzing oxidation or dehydrogenation refers to any enzyme that catalyzes oxidation or dehydrogenation.
  • the enzyme recognises and uses e.g. the compound of formula (II) as a substrate.
  • Combinations of enzymes, multiunit enzymes, wherein different units catalyse optionally different reactions, fused or joined enzymes, or enzymes coupled to another structural or a non-catalytic compound, unit, subunit or moiety, are also contemplated within the present invention as long as the requirement of being capable of catalyzing oxidation or dehydrogenation is fulfilled.
  • the enzyme can be for example an enzyme found in the electron transfer chain of the prokaryote or eukaryote cells or in the biochemical pathways of alcohols, aldehydes or sugars in eukaryote or prokaryote cells. Enzymes that would normally act upon natural substrates were unexpectedly found to recognise and oxidise rather complex synthetic compounds, in particular convert lactols into lactones or possibly into esters.
  • the reactions which are meant to be used for the an enzyme capable of catalyzing oxidation or dehydrogenation, do normally not occur in the nature, because the substrate is different from the natural occurring ones, like for example expempting from using the ethanol, methanol, acetaldehyde, acetic acid, naturally occurring sugars or naturally occurring amino acids, or acids obtained from the sugars, like for example gluconic acid.
  • synthetic substrates as disclosed herein even if being rather structurally complex, can yet be easily processed by using the enzyme capable of catalyzing oxidation or dehydrogenation in order to finally obtain API, particularly statin, or intermediates thereof, including e.g. a lactone compound of formula (I).
  • enzyme can be chosen from oxydoreductases.
  • the enzyme applicable in the present invention belongs primarily to EC 1.1 (oxidoreductases acting predominantly on the CH-OH group of donors), more specifically to, but not limited to subclasses: EC 1.1.1 (with NAD + or NADP + as acceptor), EC 1.1.2 (with a cytochrome as acceptor), EC 1.1.3 (with oxygen as acceptor), EC 1.1.5 (with a quinine or flavine or similar compound as acceptor). Any of the oxidoreductases known in the art may be used for the reaction regardless of their sequence identity.
  • Enzymes having activity of oxidation/dehydrogenation of sugars have been widely used in the industry. Typical examples of oxidative fermentations are traditional production of D-gluconate (gluconic acid), L-sorbose and others. These processes were developed as a practical industry based on empirically found properties of some microorganisms before the clarification of the molecular mechanisms of the responsible enzymes [Adachi, 2007].
  • Sugar oxidising enzymes had been used in food processing as additives, in dairy and the lactoperoxidase system for food preservation, in breadmaking, for producing dry egg powder, as antioxidants/preservatives (oxygen scavengers), for reducing alcohol wine, as glucose assays and fuel cells [Wong, 2008]. Sugar oxidising enzymes had been used also as amperometric biosensors, e.g.
  • a well known enzyme capable of oxidation of six-membered sugars is Glucose oxidase, Gox (EC 1.1.3.4), which is commercially available from Sigma as an extract from Aspergillus niger. This enzyme has a very narrow substrate specificity [Keilin, 1952]. It is produced naturally in some fungi and insects where its catalytic product, hydrogen peroxide, acts as an anti-bacterial and anti-fungal agent. Gox is generally regarded as safe, and Gox from A . niger is the basis of many industrial applications [Wong, 2008]. Gox-catalysed reaction has also been used in baking, dry egg powder production, wine production, gluconic acid production, etc.
  • Glucose oxidase is capable of oxidising monosaccharides, nitroalkanes and hydroxyl compounds [Wilson, 1992].
  • reaction rate of glucose 100 %
  • 2-deoxy-D-glucose 20-30 %
  • 4-O-methyl-D-glucose 15 %
  • 6-deoxy-D-glucose 10 %
  • the activities of glucose oxidase against other substrates are typically poor, with reaction rates lower than 2 % of glucose's [Keilin, 1948; Pazur, 1964; Leskovac, 2005].
  • the enzyme capable of catalyzing oxidation or dehydrogenation is a dehydrogenase.
  • the enzyme is capable of catalyzing sugar dehydrogenation, more particularly aldose dehydrogenation.
  • the enzyme is sugar dehydrogenase (EC 1.1).
  • the enzyme is an aldose dehydrogenase or a glucose dehydrogenase.
  • substrate specificity and capability of the enzyme to catalyse the oxidation/dehydrogenation of compound (II) or other compounds contemplated herein are independent of terminology found in the current art.
  • the natural substrate for the sugar dehydrogenases are various sugars that get oxidized.
  • the broad range of sugars that aldose dehydrogenase can act upon encompasses pentoses, hexoses, disaccharides and trisaccharides.
  • the enzyme capable of catalyzing oxidation or dehydrogenation is specific for oxidation at position C1. In the case of the aldose 1-dehydrogenase the enzyme oxidizes the aldehyde or cyclic hemiacetal to lactone.
  • sugar oxidoreductases are divided into classes, according to electron acceptors (in some cases these are the cofactors these enzymes use in order to become functional, i.e. FAD, NAD(P) + or PQQ).
  • FAD electron acceptor
  • NAD(P) + or PQQ electron acceptor
  • PQQ dependent dehydrogenases EC 1.1.5 are preferred according to the present invention.
  • FAD- (flavoprotein dehydrogenases) and PQQ-dependent sugar dehydrogenases EC 1.1.5, use flavin adenine dinucleotide (FAD) or pyrroloquinoline quinine (PQQ) cofactors respectively, and are located on the outer surface of the cytoplasmic membrane of bacteria, facing the periplasmic space with their active sites. These are often termed membrane sugar dehydrogenases. Alternatively, especially in the PQQ-dependent sugar dehydrogenases group, many enzymes are found in soluble form, located in the periplasmic space.
  • FAD flavin adenine dinucleotide
  • PQQ pyrroloquinoline quinine
  • the electrons generated by the oxidation process are transferred from substrates via the enzyme cofactor (electron carrier) to the terminal ubiquinol oxidase with ubiquinone as a mediator in the respiratory chain of host organisms.
  • the final acceptor of electrons is oxygen which is reduced to water by the respiratory chain oxidoreductases.
  • a respiratory chain is a series of oxidoreductive enzymes, having ability to transfer electrons from a reduced molecule in a cascade of finely tuned stepwise reactions, which are concluded by reduction of oxygen. Each step uses a difference in redox potential for useful work, e.g. transfer of protons across the cytoplasmic membrane, reduction of other molecules etc. Electron carriers have a major role in the respiratory chain as well as in overall cell's oxidoreductive processes.
  • Electrons can enter the respiratory chain at various levels. At the level of a NADH dehydrogenase which oxidizes NADH/NADPH (obtained by various oxidoreductive processes in the cell) with transfer of electrons to ubiquinone pool and release of protons to extracellular space.
  • NADH dehydrogenase which oxidizes NADH/NADPH (obtained by various oxidoreductive processes in the cell) with transfer of electrons to ubiquinone pool and release of protons to extracellular space.
  • oxidoreductases can transfer electrons directly to ubiquinon via enzyme bound cofactors (FAD,PQQ).
  • the ubiquinoles are furher oxydised by terminal oxidoreductases such as Cytochomes, Nirate reductsases etc., whereby the electrons are coupled with intracellular protons to reduce oxygen (forming water) and ubiquinole bound protons are released into extracellular space.
  • Any system which is capable of translocation of protons exploiting redox potential is ofted known as a proton pump.
  • a cross membrane proton potential is thereby established and is the driving force for function of ATP synthases. These levels have successively more positive redox potentials, i.e. successively decreased potential differences relative to the terminal electron acceptor.
  • Individual bacteria often simultaneously use multiple electron transport chains. Bacteria can use a number of different electron donors, a number of different dehydrogenases, different oxidases and reductases, and different electron acceptors.
  • E.g., E . coli when growing aerobically using glucose as an energy source) uses two different NADH dehydrogenases and two different quinol oxidases, for a total of four different electron transport chains operating simultaneously.
  • an oxidoreductase for example sugar dehydrogenase
  • a electron acceptor for example sugar dehydrogenase
  • an oxidoreductase for example sugar dehydrogenase
  • an electron acceptor for example sugar dehydrogenase
  • this is provided by the respiratory chain
  • artificial electron acceptors with appropriate redox potential compared to substrate/enzyme/cofactor cascade can be used.
  • the disadvantage is that the artificial electron acceptor has to be provided in rather large quantities, in other words normally in equimolar amount to the substrate being oxidized.
  • the acceptor of electrons generated by the enzyme capable of catalyzing oxidation or dehydrogenation may be provided in the reaction mixture in order to promote electron flow and the oxidation or dehydrogenation of compound (II).
  • the acceptor may be selected from but is not limited to: dichlorophenolindophenol (DCPIP), phenazine methosulfate (PMS), potassium ferricyanide (PF), potassium ferrioxalate, p-benzoquinone, phenyl-p-benzoquinone, duroquinone, silicomolybdate, vitamin K3, diaminodurene (DAD), N,N,N',N -tetramethyl- p -phenylenediamine (TMDP).
  • Electron acceptor may also be oxygen.
  • a person skilled in art will recognize and can e.g. use compounds listed as Hill reagents, dyes that act as artificial electron acceptors, changing colors when reduced, and find many additional candidate acceptors from literature
  • the preferred aspect of this invention deals with PQQ dependent dehydrogenases (quinoproteins, EC 1.1.5), more specifically PQQ dependant sugar 1-dehydrogenases (EC 1.1.5.2).
  • Ylil is aldose sugar dehydrogenase from E. coli, which requires PQQ for its activity [Southall, 2006]. While E. coli lacks the ability to synthesize PQQ itself [Hommes, 1984; Matsushita, 1997], it shows positive chemotaxis effect towards PQQ, found in environment [de Jonge, 1998], and can use an externally supplied cofactor [Southall, 2006].
  • Ylil aldose sugar dehydrogenase is a soluble, periplasmic protein, containing N-terminal signal sequence necessary for translocation into the periplasm through the cytoplasmic membrane.
  • Ylil aldose sugar dehydrogenase (Asd) fold contains six four stranded antiparallel ⁇ -sheets with PQQ-binding site lying on the surface of the protein in a shallow, solvent exposed cleft.
  • Ylil protein is a monomer, each binding two calcium ions, one of them lying in the PQQ binding pocket, and the other compressed between two of the six strands that make up the propeller fold [Southall, 2006].
  • E. coli contains also membrane-bound glucose dehydrogenase (mGDH), which is also a quinoprotein involved within the respiratory chain in the periplasmic oxidation of alcohols and sugars [Yamada, 2003].
  • mGDH membrane-bound glucose dehydrogenase
  • Acinetobacter calcoaceticus GDH Another example of PQQ dependant sugar dehydrogenase is Acinetobacter calcoaceticus GDH.
  • Acinetobacter calcoaceticus GDH At least two distinct quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus are known: as usual membrane-bound form (mGDH) and as soluble form (sGDH), which contains a 24-amino-acid N-terminal signal sequence needed for translocation through the cytoplasmic membrane into the periplasm. Both forms are different in all characteristics, e.g. substrate specificity, molecular size, kinetics, optimum pH, immunoreactivity.
  • sGdh oxidizes preferably D-glucose, maltose and lactose and less successfully D-fucose, D-xylose, D-galactose, while mGdh is less reactive with disaccharides; it oxidises preferably D-glucose, D-fucose, D-xylose, D-galactose, D-ribose, yet less successfully maltose, and lactose [Adachi, 2007].
  • the two possible reaction mechanisms for s-GDH are: (A) The addition-elimination mechanism comprises general base-catalyzed proton abstraction followed by covalent addition of the substrate and subsequent elimination of the product. (B) Mechanism comprising general base catalyzed proton abstraction in concert with direct hydride transfer from substrate to PQQ, and tautomerization to PQQH 2 [Oubrie, 1999]. A similar mechanism is assumed to be the case for E.coli Ylil aldose dehydrogenase enzyme.
  • the sGDH also requires calcium for dimerization and function [Olsthoorn, 1997].
  • the present structures confirm the presence of three calcium binding sites per monomer [Oubrie, 1999].
  • D-gluconate production ( Gluconobacter oxydans ) in classic fermentation processes as well as production of various natural sugars.
  • Gluconobacter oxydans organism is well known for its important ability to incompletely oxidize natural carbon substrates such as D-sorbitol (producing L-sorbose for vitamin C synthesis), glycerol (producing dihydroxyacetone), D-fructose, and D-glucose (producing gluconic acid, 5-keto-, 2-keto- and 2,5-diketogluconic acid) for the use in biotechnological applications [Gupta, 2001].
  • PQQ dependent dehydrogenases are found in Acinetobacter calcoaceticus, an industrial microorganism used in vinegar production.
  • PQQ dependent sugar dehydrogenases may have found its use also in nanotechnology as biofuel cells [Gao, 2010]. Soluble PQQ dependent glucose dehydrogenases have become the major group of enzymes used in biosensor systems for self monitoring of blood glucose, because these enzymes, unlike glucose oxidase, are independent of oxygen presence [Heller, 2008].
  • the skilled person will become aware and derive how to select the enzyme capable of catalyzing oxidation or dehydrogenation in order to convert e.g. the compound of formula (II) to the compound of formula (I) based on its substrate specificity or promiscuity, operational pH, temperature and ionic strength window, a need of additional ions or cofactors, or the like.
  • Substrates and reaction conditions are normally chosen to give the optimal activity of the enzyme.
  • the substrates and conditions to provide the least inhibitory effect on the cell that hosts the enzyme, or deteriorate stability of the product can be leveraged against the substrates and conditions by which the optimal activity is reached.
  • reaction conditions include in one aspect that the temperature, pH, solvent composition, agitation and length of the reaction allow accumulation of the desired product.
  • the skilled person will know with the disclosure provided herein to adapt the conditions in terms of applying proper pH, temperature and reaction time to prevent the product, e.g. lactone or ester, to deteriorate.
  • specific cofactors, co-substrates and/or salts can be added to the enzyme in order to either allow or improve its activity.
  • Cofactors are salts or chemical compounds.
  • said species are already included in the solvent mixture, especially if the enzyme is comprised within living whole cell, inactivated whole cell, homogenized whole cell, or cell free extract. Nevertheless, the cofactors, co-substrates and/or salts can be further added to the enzyme, solvent or reaction mixture.
  • cupric, ferric, nickel, selenium, zinc, magnesium, calcium, molybdenum, or manganese ions, or nicotinamid adenine dinucleotide (NAD), nicotinamid adenine dinucleotide phosphate (NADP+), lipoamide, ascorbic acid, flavin mononucleotide, flavin adenine dinucleotide (FAD), coenzyme Q, coenzyme F420, pyrroloquinoline quinine, coenzyme B, glutathione, heme, tetrahydrobiopterin, or the like can be added to the enzyme, to the solvent or medium or to the reaction mixture comprising the enzyme.
  • aldose-1-dehydrogenase or preferably Ylil
  • calcium ions and pyrroloquinoline quinine or similar electron acceptor is added to the reaction mixture, enzyme or solvent or medium.
  • suitable conditions are exemplified in the examples hereinafter.
  • a dehydrogenase for use in the present invention may be particularly chosen among any enzyme that has oxidative activity towards above substrate (II).
  • any sugar 1-dehydrogenase known in the art can be used regardless of their sequence identity to the enzymes listed below, notably dehydrogenases.
  • the enzyme oxidizes the aldehyde or cyclic hemiacetal to lactone.
  • Special variants of the enzymes like for example enzymes found in the termoresistant microorganism strains, are also contemplated within the present invention.
  • GDH 01 is a dehydrogenase encoding gene comprised within nucleotide sequence of SEQ ID NO. 01 or an amino acid sequence of SEQ ID NO. 02.
  • GDH 02 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 03 or an amino acid sequence of SEQ ID NO. 04.
  • GDH 03 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 05 or an amino acid sequence of SEQ ID NO. 06.
  • GDH 04 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 07 or an amino acid sequence of SEQ ID NO. 08.
  • GDH 05 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 09 or an amino acid sequence of SEQ ID NO. 10.
  • GDH 06 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 23 or an amino acid sequence of SEQ ID NO. 24.
  • GDH 07 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 25 or an amino acid sequence of SEQ ID NO. 26.
  • GDH 08 is a dehydrogenase having a nucleotide sequence of SEQ ID NO.
  • GDH 09 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 29 or an amino acid sequence of SEQ ID NO. 30.
  • GDH 10 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 31 or an amino acid sequence of SEQ ID NO. 32.
  • GDH 11 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 33 or an amino acid sequence of SEQ ID NO. 34.
  • GDH 12 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 35 or an amino acid sequence of SEQ ID NO. 36.
  • GDH 13 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 37 or an amino acid sequence of SEQ ID NO. 38.
  • GDH 14 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 39 or an amino acid sequence of SEQ ID NO. 40.
  • GDH 15 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 41 or an amino acid sequence of SEQ ID NO. 42.
  • GDH 16 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 43 or an amino acid sequence of SEQ ID NO. 44.
  • GDH 17 is a dehydrogenase having a nucleotide sequence of SEQ ID NO.
  • GDH 18 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 47 or an amino acid sequence of SEQ ID NO. 48.
  • GDH 19 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 49 or an amino acid sequence of SEQ ID NO. 50.
  • GDH 20 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 51 or an amino acid sequence of SEQ ID NO. 52.
  • GDH 21 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 53 or an amino acid sequence of SEQ ID NO. 54.
  • GDH 22 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 55 or an amino acid sequence of SEQ ID NO. 56.
  • GDH 23 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 57 or an amino acid sequence of SEQ ID NO. 58.
  • GDH 24 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 59 or an amino acid sequence of SEQ ID NO. 60.
  • GDH 25 is a dehydrogenase having a nucleotide sequence of SEQ ID NO. 61 or an amino acid sequence of SEQ ID NO. 62.
  • a sugar dehydrogenase for use in the present invention may be any compound that has sugar 1- dehydrogenase activity toward compound (II).
  • the sugar 1. dehydrogenase is a PQQ dependat sugar 1- dehydrogenase.
  • sutable PQQ dependant sugar 1-dehydrogenase examples include but are not limited to: GDH 01, GDH 02, GDH 03, GDH 04, GDH 05, GDH 06, GDH 07, GDH 08, GDH 09, GDH 10, GDH 11, GDH 12, GDH 13, GDH 14, GDH 15, GDH 16, GDH 17, GDH 18, GDH 19, GDH 20, GDH 21, GDH 22, GDH 23, GDH 24 and GDH 25, wherein each enzyme is identified by it's corresponding nucleotide sequence or respective codon optimized nucleotide sequence or aminoacid sequence as set forth in sequence listing above.
  • the present invention provides sugar dehydrogenases having an amino acid sequence identitiy of at least 50 % thereof; preferably at least 70 % thereof, to any of dehydrogenases selected from GDH 01, GDH 02, GDH 03, GDH 04, GDH 05, GDH 06, GDH 07, GDH 08, GDH 09, GDH 10, GDH 11, GDH 12, GDH 13, GDH 14, GDH 15, GDH 16, GDH 17, GDH 18, GDH 19, GDH 20, GDH 21, GDH 22, GDH 23, GDH 24 and GDH 25.
  • the amino acid sequence identities are determined by analysis with sequence comparison algorithm or by visual inspection.
  • the sequence comparison IS made with default settings in AlignX module, component of Vector NTI Advance 11.0 software (Invitrogen), using clustal W algoritm at default settings.
  • a preferable sugar 1-dehydrogenase provided by this invention may be the sugar dehydrogenase originating from Escherichia coli identified as GDH01 in the above sequence listing and having corresponding nucleotide sequence SEQ ID NO. 01 and an amino acid sequence of SEQ ID NO. 02.
  • Equaly preferable sugar 1-dehydrogenase provided by this invention may be the sugar dehydrogenase originating from Escherichia coli identified as GDH02 in the above sequence listing and having corresponding nucleotide sequence SEQ ID NO. 03 and an amino acid sequence of SEQ ID NO. 04.
  • Another preferable sugar 1-dehydrogenase provided by this invention may be selected from the sugar dehydrogenase originating from Acinetobacter calcoaceticus coli identified as GDH03 in the above sequence listing and having corresponding nucleotide sequence SEQ ID NO. 05 and an amino acid sequence of SEQ ID NO. 06.
  • Yet another preferable sugar 1-dehydrogenase provided by this invention may be selected from the modified sugar dehydrogenase originating from Acinetobacter calcoaceticus coli identified as GDH04 in the above sequence listing and having corresponding nucleotide sequence SEQ ID NO. 07 and an amino acid sequence of SEQ ID NO. 08.
  • the most preferable sugar 1-dehydrogenase provided by this invention may be the sugar dehydrogenase from originating from Escherichia coli identified as GDH01 in the above sequence listing and having corresponding nucleotide sequence SEQ ID NO. 01 and an amino acid sequence of SEQ ID NO. 02.
  • the said sugar 1-dehydrogenase is also described in the art and within this invention as PQQ dependant sugar dehydrogenase, PQQ dependant glucose dehydrogenase, PQQ dependant aldose dehydrogenase, aldose dehydrogenase, aldose dehydrogenase quinoprotein or glucose dehydrogenase quinoprotein. This particular enzyme is encoded by gene ylil naturally occurring in E .
  • the present invention illustratively makes use of sugar dehydrogenases having an amino acid sequence identitiy of at least 21,8% thereof; 50 % thereof; preferably at least 70 % thereof, to the aminoacid sequence SEQ ID NO. 02.
  • Oxidation/dehydrogenization activity towards compound (II) may be screened among different microorganisms and/or enzymes.
  • the term "analysis material” as used herein refers to any microorganism and/or enzyme that can be used in screening method to screen for and identify microorganism and/or enzyme able to convert compound (II) to compound (I) as the living whole cell catalyst, resting whole cell catalyst, cell free lysate, partially purified or purified enzyme, immobilized enzyme or any other form of catalyst as provided by this invention of any microorganism regardless of it being native or genetically modified microorganism.
  • a term “analysed material” includes all preparations of candidate catalyst as described above.
  • “analysed material” may be obtained having regard to its cultivation properties. Cultivation may be performed to obtain biomass of "analysed material” in growth medium which satisfies the nutrient needs. Cultivation may be performed in liquid medium or on solid medium. Growth medium and conditions of cultivating may be chosen from but are not limited to Difco & BBL Manual, 2010 and to other protocols well known to person skilled in the art. Cultivated microorganisms may be prepared in different forms of catalyst as provided by this invention. In particular "analysed material” is brought in contact with compound (II) in such conditions that allow forming and accumulation of compound (I).
  • These conditions include in one aspect that the " analysesd material” is provided at sufficient load to be able to perform the oxidationldehydrogenization, in another aspect that the substrate and electron acceptors are present in the reaction in an amount that displays minimal inhibition of the activity of the catalyst, in another aspect that the temperature, pH, solvent composition, agitation and length of reaction allow accumulation of desired product, in another aspect that said conditions do not have detrimental effect on product stability.
  • the conditions may be defined as indicated by, or as modified or varied from, values or conditions disclosed in examples.
  • “Analysed material” may be able to intrinsically provide all cofactors needed for activity towards compound (I) (naming PQQ), or "analysed material” possess the capability of converting compound (II) to compound (I) when PQQ is provided externally as described in this invention.
  • All screening methods provided herein to PQQ may preferebly be added concentrations described and exemplified. It will become apparent to a person skilled in the art that quantification of PQQ presence can be determined by the above method and as described elsewhere in the present specification. In this case the material used for the method has to be depleted of PQQ or contain no PQQ already by it's nature.
  • One such method for screening of and identifying candidate catalysts is to bring into contact the "analysis material" with a compound (II). Converting of compound (II) to compound (I) should be performed at optimal reaction conditions as described above. Detection of substrates converting to product in presence of "analysis material” can be achieved by any of the well known chromatographic methods known in the art. The non-limiting examples include liquid HPLC, GC, TLC analysis etc.
  • An exemplified but not limiting method for monitoring compound (I) and corresponding compound (II) is gas chromatography analysis (chromatographic column: DB-1 100 % dimethylpolysiloxane; temperature program: initial temperature: 50 °C, initial time: 5 min, temperature rate: 10 °C/min, final temperature: 215 °C, final time: 10 min; injector: split/spliteess injector; carrier gas: helium, initial flow: 10 mL/min; detector: flame ionization detector (FID), detector temperature: 230 °C).
  • the prerequisite for carrying out such method is a presence of electron acceptor in the reaction mixture.
  • Another screening method provided by this invention is a method performed in presence of alternative artificial electron acceptors with appropriate redox potential compared to a substrate/enzyme/cofactor cascade that can be used.
  • the present invention provides a screening method using artificial electron acceptor which changes its optically measurable property or properties (such as color, absorbance spectra, etc.) when reduced.
  • artificial electron may be provided in the reaction mixture (a dye-linked system) in order to promote electron flow, hence being indicative of the oxidation or dehydrogenation of compound (II).
  • the acceptor/indicator may be selected from but is not limited to: 2,6-dichlorophenol indophenol (DCPIP), phenazine methosulfate (PMS), potassium ferricyanide (PF), potassium ferrioxalate, p-benzoquinone, phenyl- p -benzoquinone, duroquinone, silicomolybdate, vitamin K3, diaminodurene (DAD), N,N,N',N' -tetramethyl- p- phenylenediamine (TMDP).
  • DCPIP 2,6-dichlorophenol indophenol
  • PMS phenazine methosulfate
  • PF potassium ferricyanide
  • TMDP N,N,N',N' -tetramethyl- p- phenylenediamine
  • DCPIP and PMS are used in concentrations from about 0.01 mM to about 10mM for both said artificial electron acceptors.,in particular from about 0.05mM to about 5mM DCPIP combined with 0.01 mM to about 2mM DCPIP.
  • the amount of DCPIP in a screening method is provided in concentration from 0.1mM to about 1mM combined with PMS in concentration from 0.05mM to about 0.5 mM.
  • the DCPIP combined with PMS is provided in the amount which allows observation of reduction of absorbance in timeline that can be spectrophotometrically followed.
  • the compound (II) may be dissolved in appropriate aqueous solution and used in a screening method in concentrations from about 0.5 mM to about 1 M preferably from about 10 mM to about 500 mM, most preferably 20 mM to 200 mM.
  • Compound (II) may be dissolved in distilled water or in suitable buffered solution. Suitable buffers for adjusting pH value are made with acids, bases, salts or mixtures thereof in particular phosphoric acid and sodium hydroxide may be used.
  • the aqueous suspension, in which the screening method is performed, may be buffered to pH 5.5 to 9.0, preferably to 6.0 to 8.5, more preferably 7.0. to 8.0.
  • “Analysed material” is added to reaction mixture in the said aqueous suspension (particularly in a concentration range from about 0.05 g/L to about 50 g/L), optionally in buffered solution (in particularly in phosphate buffer pH 6.0 to 8.5). Screening and identifying of catalysts capable of converting compound (II) to compound (I) can be observed spectrophotometrically following absorbance reduction in time line, may be at wavelength between 380 nm and 750 nm, perferably at wavelength between 450 nm and 650 nm, more preferably between 550 nm and 650 nm.
  • the aldose dehydrogenase activity unit is defined as absolute value of reduction in absorbance unit per minute per wet weight of cultured microorganisms used for preparation of any "analysis material" (abs[mAU min -1 mg -1 ]).
  • abbreviations of cells per minute per wet weight of cultured microorganisms used for preparation of any "analysis material" abbreviations [mAU min -1 mg -1 ]).
  • cell density of tested microorganisms may be quantified as wet weight in mg per mL of sample, protein concentrations and/or other indirect or direct methods for quantification well known to person skilled in the art.
  • Pyrroloquinoline quinine (4,5-dihydro-4,5-dioxo-1H-pyyrolo-[2,3-f]quinoline-2,7,9-tricarboxylic acid: PQQ) is a molecule needed for functioning when using quinoproteins.
  • PQQ a redox cofactor, which is water soluble and heat-stable, is considered as the third type of coenzyme, after nicotinamide and flavin in biological oxidoreductions and was discovered by Hauge, 1964. To that time unknown redox cofactor was also found by Anthony and Zatman in alcohol dehydrogenase and was named by them as methoxantin [Anthony, 1967].
  • PQQ has been found in both prokaryotic (such as Klebsiella pneumoniae, Acinetobacter calcoaceticus, Methylobacterium extorquens, Kluyvera intermedia, Gluconobacter oxydans, Pseudomonas aeruginosa, Erwinia amylovora, Rahnella aquatilis, Deinococcus radiodurans) and eukaryotic organisms (such as Polyporus versicolor , Rhus vernicifera ) [Goodwin, 1998; Hoelscher, 2006; Yang, 2010]
  • prokaryotic such as Klebsiella pneumoniae, Acinetobacter calcoaceticus, Methylobacterium extorquens, Kluyvera intermedia, Gluconobacter oxydans, Pseudomonas aeruginosa, Erwinia amylovora, Rahnella aquatilis, Deinococcus radiodurans
  • a generally accepted structure of PQQ is: wherein the PQQox is the oxidized form of the cofactor and PQQred is the reduced from of the cofactor.
  • the number of genes involved in biosynthesis of PQQ varies between species, but in general it is known that for biosynthesis at least five or six genes are needed, usually clustered in the pqq ABCDE or pqqABCDEF operon.
  • the number and organization of the genes is variable as it can be seen in following examples.
  • Klebsiella pneumoniae the PQQ biosynthetic genes are clustered in the pqqABCDEF operon, while in Pseudomonas aeruginosa the pqqF is separated from the pqqABCDE operon.
  • Acinetobacter calcoaceticus there is a pqqABCDE but no pqqF gene is known.
  • a facultative methylotroph Methylobacterium extorquens AM1 contains a pqqABC / DE operon in which the pqqC and pqqD genes are fused, while the pqqFG genes form an operon with three others genes.
  • PQQQ protein kinase
  • backbone of PQQ is constructed from glutamate and tyrosine. Most probably these amino acids are encoded in the precursor peptide PqqA.
  • the length of the small peptide varies between different organisms (from 23 amino acids in K. pneumoniae to 39 in P. fluorescens, respectively) and in all variants in the middle of the PqqA peptide motif Glu-X-X-X-Tyr is conserved.
  • the PqqB protein might be involved in its transportation into the periplasm and thus is not directly required for PQQ biosynthesis.
  • PqqC protein Residues of PqqC protein are highly conserved within PqqC proteins, which are responsible for catalyzing the final step in PQQ formation, from different bacteria. Although the alignment of protein sequences of PqqD proteins from different organisms shows strictly conserved residues, the function of PqqD is not fully resolved. In Klebisella pneumoniae it was shown that PqqE recognizes the PqqA, which links the C9 and C9a, afterwards it is accepted by PqqF which cuts out the linked amino acids. In the said organism it was shown that the next reaction (Schiff base) is spontaneous, following dioxygenation. The last cyclization and oxidation steps are catalysed by PqqC [Puehrunger, 2008].
  • PQQ gene clusters comprising only pqqABCDE genes and lacking pqqF may be used to provide compete PQQ biosynthetic maschinery in E . coli .
  • the pitrilysin protease encoded by tldD gene
  • tldD gene is apparently complementig for the activity of pqqF gene found in some microorganisms.
  • E . coli lacks the ability to synthesize PQQ itself [Hommes, 1984; Matsushita, 1997], it shows positive chemotaxis effect towards PQQ, found in environment [de Jonge, 1998], and can use an externally supplied cofactor [Southall, 2006].
  • PQQ biosynthesis genes could be recombinantly expressed in E . coli , what is one of the aspects described in this invention.
  • the host organism for the production of appropriate dehydrogenase has intrinsic PQQ biosynthetic capability, in other words, contains functional genes for PQQ biosynthesis already integrated in its genetic material.
  • PQQ biosynthetic capability in other words, contains functional genes for PQQ biosynthesis already integrated in its genetic material.
  • Non-limiting examples are: Klebsiella pneumoniae , Acinetobacter calcoaceticus , Methylobacterium extorquens , Kluyvera intermedia , Gluconobacter oxydans , Pseudomonas aeruginosa , Erwinia amylovora , Rahnella aquatilis, Deinococcus radiodurans and others.
  • the present invention provides microorganisms with native ability to produce PQQ that can be used as hosts for homolohous or heterologous expression of PQQ dependant aldose dehydrogenases.
  • Said microorganisms are preferably selected among bacteria, more preferably industrially culturable bacteria and particularly from Klebsiella pneumoniae, Acinetobacter calcoaceticus, Methylobacterium extorquens, Kluyvera intermedia , Enterobacter , Gluconobacter oxydans , Pseudomonas aeruginosa , Erwinia amylovora , Rahnella aquatilis , Deinococcus radiodurans .
  • Klebsiella pneumoniae Acinetobacter calcoaceticus, Pseudomonas aeruginosa, Erwinia amylovora, Gluconobacter oxydans may be used.
  • the present invention provides microorganisms with natural capability to convert compound (II) to compound (I). No genetic modifications are needed with provided organisms in order to obtain a catalyst capable of performing the desired oxidation. Therefore this invention provides microorganisms for the presently disclosed purpose and use, selected among bacterial origin, more particularly from genera: Klebsiella Enteorobacter, Acinetobacter, Rhizobioum. Methylobacterium, Kluyvera, Gluconobacter, Pseudomonas, Erwinia, Rahnella and Deinococcus.
  • the microorganisms may be selected from Klebsiella pneumoniae , Acinetobacter calcoaceticus, Methylobacterium extorquens, Kluyvera intermedia, Enterobacter, Gluconobacter oxydans, Pseudomonas aeruginosa, Erwinia amylovora, Rahnella aquatilis, Deinococcus radiodurans , most preferably from: Gluconobacter oxydans , Acinetobacter calcoaceticus and Kluyvera intermedium.
  • microorganisms with desired properties to carry out this invention. Further, methods are disclosed and provided which allow screening for and identification of such microorganisms.
  • the third option is especially applicable to microorganisms which do not have intrinsic capability of biosynthetis of PQQ, such as Escherichia coli. It is well known in the art that some microorganims such as E. coli and most of higher organisms have PQQ-dependent enzymes encoded in their genomes and expresses in certain conditions but lack biosynthesis of PQQ [Matshushita, 1997]. It is contemplated in the art that such microorganisms obtain the PQQ as an essential nutrient, or with other words, a vitamin. Ways to establish biosynthesis of PQQ in such organisms to be used for the present invention will be apparent to a person skilled in the art.
  • Non-limiting examples of microorganims suitable for this purpose include Klebsiella pneumonia, Methylobacterium extorguens, Pseudomonas aeruginosa, Gluconobacter oxydans, Kluyvera intermedia, Erwinia amylovora and others.
  • a term "heterologous expression of PQQ gene cluster" will be immediately understood by a person skilled in the art, as a well established term describing the above procedures.
  • any PQQ gene cluster may be used, providing that said gene cluster encodes functional proteins as described above with capability of biosynthesis of PQQ either alone or in concert with the host organism's enzymes.
  • the pQQ gene cluster can be obtained from any living organism producing PQQ.
  • the PQQ gene cluster can be obtained from any microorganisms selected among bacterial, more particularly from genera: Klebsiella Enteorobacter, Acinetobacter, Rhizobioum, Methylobacterium,Kluyvera, Gluconobacter, Pseudomonas, Erwinia, Rahnella and Deinococcus.
  • the microorganisms may be selected from Klebsiella pneumoniae, Acinetobacter calcoaceticus, Methylobacterium extorquens, Kluyvera intermedia, Enterobacter, Gluconobacter oxydans, Pseudomonas aeruginosa, Erwinia amylovora, Rahnella aquatilis, Deinococcus radiodurans, most preferably from: Gluconobacter oxydans, Acinetobacter calcoaceticus and Kluyvera intermedia.
  • PQQ gene clusters are included, but are not limited to nucleotide sequences of clusters or included genes in the sequence list, which are identified by their nucleotide sequences or amino acid sequences set forth in sequence listings.
  • any of the PQQ clusters providing functional genes known in the art may be used for the reaction regardless of their sequence identity to the listed PQQ clusters, genes comprised within and proteins encoded by said genes.
  • PQQ 01 is a PQQ encoding gene cluster from Gluconobacter oxydans 621H comprised within nucleotide sequence of SEQ ID NO. 68 and allows expression of genes pqqA, pqqB, pqqC, pqqD and pqqE encoding proteins PqqA, PqqB, PqqC, PqqD and PqqE with aminoacid sequence SEQ ID NO. 12, 13, 14, 15, 16, respectively.
  • PQQ 02 is a PQQ encoding gene cluster from Kluyvera intermedia comprised within nucleotide sequence of SEQ ID NO. 69 and allows expression of genes pqqA, pqqB, pqqC, pqqD and pqqE encoding proteins PqqA, PqqB, PqqC, PqqD and PqqE with aminoacid sequence SEQ ID NO. 18, 19, 20, 21, 22, respectively.
  • PQQ 03 is a gene cluster pqqABCDEF from Klebsiella pneumoniae 324 having a nucleotide sequence of SEQ ID. NO 63. and allows expression of genes pqqA, pqqB, pqqC, pqqD, pqqE and pqqF encoding proteins PqqA, PqqB, PqqC, PqqD, PqqE and PqqF.
  • accession number CP000964 at NCBI genome database having location between 2602846 and 2599706.
  • PQQ 04 is a gene clusters pqqABC / DE and pqqFG from Methylobacterium extorguens AM1 having nucleotide sequences of SEQ ID. NO 64 and SEQ ID. NO 65, respectively and allows expression of genes pqqA, pqqB, pqqC, pqqD, pqqE and pqqF encoding proteins PqqA, PqqB, PqqC, PqqD, PqqE and PqqF.
  • accession number CP001510 at NCBI genome database having location between 1825235 and 1821763 ( pqqABC / DE ), 2401055 and 2403792 ( pqqEF ) .
  • PQQ 05 is a gene clusters pqqABCDE and pqqF from Pseudomonas aeruginosa PA7 having nucleotide sequences of SEQ ID. NO 66 and SEQ ID. NO 67, respectively and allows expression of genes pqqA, pqqB, pqqC, pqqD, pqqE and pqqF encoding proteins PqqA, PqqB, PqqC, PqqD, PqqE and PqqF.
  • accession number CP000744 at NCBI genome database having location between 3420385 and 3423578 ( pqqABCDE ), 3439512 and 3437221 ( pqqF).
  • PQQ 06 is a gene cluster pqqABCDEF from Erwinia amylovora ATCC 49946 having a nucleotide sequence of SEQ ID. NO 70. and allows expression of genes pqqA, pqqB, pqqC, pqqD, pqqE and pqqF encoding proteins PqqA, PqqB, PqqC, PqqD, PqqE and PqqF.
  • accession number FN666575 at NCBI genome database having location between 597604 and 600850.
  • a person skiled in art would also recognize additional candidate gene clusters providing for PQQ synthesis, in publicly available databases (GenBank, Swiss-Prot/TrEMBL, RCSB PDB, BRENDA, KEGG, MetaCyc) using well established data mining tools.
  • the method for measuring activity of PQQ dependant aldose dehydrogenase provided by the present invention can be used, as exemplified by the invention herein, to screen for and identify organisms capable of producing PQQ regardless of their origin (native or genetically modified), and in addition allows, if desired, a semi quantitative method for estimating the quantity of produced PQQ.
  • a PQQ dependant aldose dehydrogenase in any form, preferably expressed in E. coli (or any other microorganism unable to produce PQQ), can be used for reconstitution of active holo-enzyme.
  • a calibration curve obtained by measuring activity of said PQQ dependant aldose dehydrogenase, supplemented with various quantities of PQQ, is compared to the activity of said PQQ dependant aldose dehydrogenase which was supplemented with analysed sample. Whithin the linear range of the method, the more PQQ is present in the analysed sample, the more activity is observed.
  • the PQQ gene clusters are derived from Kluyvera intermedia or Gluconobacter oxydans. Particularly gene cluster from Gluconobacter oxydans 621H comprised within nucleotide sequence of SEQ ID NO. 68 may be used. Alternatively, a particular embodiment of this invention provides use of gene cluster from Kluyvera intermedia comprised within nucleotide sequence of SEQ ID NO. 69
  • the described gene cluster can be modified by methods known in the art, for example methods described in Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3' ⁇ " Ed., Cold Spring Harbor, NY 2001, in order to allow expression of genes encoded in said cluster in E. coli. Any of numerous strains of E.
  • E. coli K12 strains such as JM109, DH5, DH10, HB101, MG4100 etc. or E. coli B strains such as BL21, Rossetta, Origami etc.
  • Genes can be introduced into the said host strain by any genetic method known in the art, for example by transfection, transformation, electroporation, conjugal transfer and others.
  • Said gene clusters may be maintained in the said host microorganism in any form known in the art, for example encoded in a autonomously replicating plasmid or integrated into host's genome.
  • Expression of the genes encoded in said gene clusters can be obtained either by utilizing the activity of native promoters controlling the expression of said genes or by replacing the promoters by promoters which may be more suitable for expression in said host microorganism. Methods for making such modifications are well known in the art.
  • the invention provides gene cluster from Gluconobacter oxydans 621H comprised within nucleotide sequence of SEQ ID NO. 68, which is carried on a autonomously replicating plasmid comprised within the host E. coli strain.
  • the genes encoded on the cluster are expressed under control of their corresponding native promoters.
  • the invention provides gene cluster from gene cluster from Kluyvera intermedia comprised within nucleotide sequence of SEQ ID NO. 69, which is carried on a autonomously replicating plasmid comprised within the host E. coli strain.
  • the genes encoded on the cluster are expressed under control of their corresponding native promoters.
  • the genes used for provision of PQQ synthesis in E. coli are pqqABCDE and function of pqqF is provided by intrinsic activity of E. coli.
  • Another such parameter is expression strength which can be controlled by temperature of cultivation, transcriptional promoter selected, codon usage in the PQQ dependent aldose dehydrogenase encoding gene, quantity of expression inducer etc.
  • Yet another such parameter are intrinsic properties of selected PQQ dependent aldose dehydrogenase such as ability to fold correctly in heterologous host, toxicity to heterologous host, resistance to the host's degrading enzymes etc. All such parameters, which are useful for enhanced activity and optimization and methods to do so, will become apparent to persons skilled in the art.
  • the present invention for example provides a particular process comprising the step of reacting a substrate (II) under dehydrogenase catalyzed oxidation conditions to form the corresponding lactone (I), wherein the dehydrogenase is selected in first embodiment from GDH 01 or GDH 02 or GDH 03 or GDH 04 or GDH 05, or any dehydrogenase having an amino acid sequence identity of at least 70 % to those, more preferably 90 % to those. In another embodiment the dehydrogenase is selected from GDH 06 or GDH 07 or GDH 08 or GDH 09 or GDH 10, or any dehydrogenase having an amino acid sequence identity of at least 70 % to those, more preferably 90 % to those.
  • this invention relates to a method of constructing and providing appropriate synthetic biological pathways, such as exemplified with E. coli as a host microorganism, wherein DERA (deoxyribose 5-phosphate aldolase), PQQ dependant dehydrogenase and PQQ biosynthetic pathway genes are expressed simultaniously.
  • DERA deoxyribose 5-phosphate aldolase
  • PQQ dependant dehydrogenase and PQQ biosynthetic pathway genes are expressed simultaniously.
  • the respiratory chain of the host organism are established and provided also.
  • Ylil aldose dehydrogenase meets all preferred features and is thus most prefered enzyme used.
  • the Ylil encompasses any aldose dehydrogenase having an amino acid sequence identity to at least 50 % of the Ylil described herein, preferably at least 70 %.
  • the amino acid sequence identities are determined by the analysis with a sequence comparison algorithm or by a visual inspection.
  • the sequence comparison algorithm is made with AlignX algorithm of Vector NTI 9.0 (InforMax) with settings set to default.
  • the present invention relates to a process of oxidation or dehydrogenation of compound (II) using an enzyme as described above, comprising the provision of microorganism or microorganism-derived material used as a living whole cell catalyst, a resting whole cell catalyst, a cell free lysate, a partially purified or purified enzyme, an immobilized enzyme or any other form of catalyst, wherein the enzyme capable of catalyzing oxidation or dehydrogenation reaction as described above is expressed in said microorganism naturally, i.e. it being the microorganism's natural property.
  • such organism when cultivated and used as catalyst in said reaction can convert compound (II) to corresponding lactone without the need for additional genetic modification of said microorganisms.
  • Said microorganism can be selected from vide diversity of bacteria as exemplified below.
  • An organism with described properties can be selected from bacteria, more particularly proteobacteria, actinomycetales, mixobacteriaceae,. More particularly said microorganism may be selected from Gamma proteobacteriaceae. Most preferably organism in this sense is selected from the group of Enterobacteriaceae, Rihzobium, Gluconobacter and Acinetobacter.
  • R5 denotes the moiety selected from the formulae (III), (IV), (V), (VI), (VII), (VIII) and (IX).
  • the compound of formula (I) obtained by the process of the present invention can be used as an intermediate for preparing a statin.
  • the skilled person will know how to put the process step of obtaining said compound according to the present invention in the context of a statin synthesis.
  • a lactone is prepared from the lactol and then coupled to the statin backbone.
  • the statin backbone containing the aldehyde side moiety is prepared, which is subsequently converted to lactol, for example by using 2-deoxyribose-5-phosphate aldolase (DERA) enzyme, and then oxidized to lactone.
  • DOTA 2-deoxyribose-5-phosphate aldolase
  • atorvastatin as an example, one can refer to schemes 2 to 4 of the WO 2006134482 .
  • an enzyme 2-deoxyribose-5-phosphate aldolase (DERA, EC 4.1.2.4) is used for preparing the compound of formula (II), which is subsequently converted to lactone by the enzyme capable of catalyzing oxidation or dehydrogenation.
  • DERA 2-deoxyribose-5-phosphate aldolase
  • Multiple wild type, variants or mutant version of DERA enzyme are know in the art, including, but not limited to, J. Am. Chem. Soc. 116 (1994), p. 8422-8423 , WO 2005/118794 or WO 2006/134482 .
  • 2-deoxyribose-5-phosphate aldolase enzyme is used for a synthetic step just preceding the step of bringing in contact the compound of formula (II) with the enzyme capable of catalyzing oxidation or dehydrogenation.
  • DERA is used to prepare the compound of formula (II) at least in part simultaneously to conversion of said compound to the compound of the formula (I) by the enzyme capable of catalyzing oxidation or dehydrogenation.
  • the enzymes necessary to catalyse the reaction of preparing the compound of formula (II) and the reaction of oxidizing said compound to formula (I) can be used within the reaction mixture, or can be added to the reaction mixture, simultaneously or subsequently, at once, intermittently or continuously.
  • the embodiment having the compound of formula (II), and thus the starting material for the reaction with the enzyme capable of catalyzing oxidation or dehydrogenation, prepared by DERA is advantageous, because this arrangement is well compatible and it allows using aqueous solvents in the preceding step and thus makes it unnecessary to purify the compound of formula (II) prior to offering it to the enzyme capable of catalyzing oxidation or dehydrogenation for conversion to lactone.
  • the preferred embodiment is thus to bring the compound of formula (II) in contact with the enzyme capable of catalyzing oxidation or dehydrogenation without prior isolation or purification of said compound.
  • the complete reaction mixture of the preceding step can be used for the subsequent reaction, which reduces the number of process steps and simplifies the process.
  • both reaction steps are performed at least in part simulateneously, preferably completely simultaneously, the toxic lactol immediately enters into the consequent reaction and is transformed to the non-toxic lactone.
  • the second reaction step typically is not a rate limiting step, as confirmed in examples hereinafter, and proceeds faster than the first step with DERA, the steady state equilibrium of the first reaction shifts in a direction of the product. This leads to reduced time for completion of the first step and thus protects DERA from being inactivated. It also protects living cells from being disrupted by high concentrations of lactol.
  • Important aspect of present invention deals with the intrinsic capability of a microorganisms to transfer electrons produced by oxidation/dehydrogenation of (II), to oxygen (a terminal electron acceptor) via its respiratory chain. This drives the reaction of enzyme catalysed oxidation / dehydrogenation of compound (II) in a whole cell system. It will be immediately apparent that the capability of acting as an electron sink is a significant and beneficial property of whole cell systems as described hereby in the invention.
  • process conditions are understood as liquid composition, temperature, pH, pressure, wherein the measurements in dynamic process are understood to be performed in a homogenous solid/liquid/gas multiphase system.
  • the enzyme capable of catalyzing oxidation or dehydrogenation, and/or the DERA enzyme i.e. respectively alone or in combination and optionally independently, can be comprised within single or multiple living whole cell(s), inactivated whole cell(s), homogenized whole cell(s), or cell free extract(s); or are respectively purified, immobilized and/or are in the form of an extracellularly expressed protein.
  • both enzymes are comprised within same living whole cell, same inactivated whole cell or same homogenized whole cell, more preferably are within same living whole cell or same inactivated whole cell, particularly are comprised within same living whole cell, because having the enzyme in a common whole cell or at least in the common inactivated whole cell, does not demand much handling with the enzyme prior it being used in the process, which reduces costs.
  • having the enzyme comprised in a living whole cell enables simple removal of the enzyme by filtration, which alleviates final purification steps at the industrial scale. In addition, it allows a reuse of the enzyme comprised within the living cell in subsequent batches.
  • Another advantage of using the enzyme in a whole cell or at least in the inactivated whole cell is possibility of providing PQQ cofactor intrinsically as described in detail above.
  • a whole cell system capable of translating 2-deoxyribose-5-phosphate aldolase (DERA) enzyme and an enzyme capable of catalyzing oxidation or dehydrogenation can be arranged to overexpress both of the genes needed for said translation. Measn for overexpression are known to the person skilled in the art, and are sometimes referred to elsewhere herein.
  • an expression system comprising one or more cell types, the respective cell type(s) being genetically engineered to express, in the totality of cell type(s), both the 2-deoxyribose-5-phosphate aldolase (DERA) enzyme and an enzyme capable of catalyzing oxidation or dehydrogenation.
  • DUA 2-deoxyribose-5-phosphate aldolase
  • An expression system can be made up of appropriate organisms or cells and optionally further factors and additives, wherein reference is made to the disclosure provided herein.
  • this invention provides a method of constructing or providing synthetic biological pathway for use in the present invention, examplified with E. coli as a host microorganism, wherein DERA (deoxyribose 5-phosphate aldolase), PQQ dependant dehydrogenase and PQQ biosynthetic pathway genes are expressed simultaniously.
  • said synthetic biological pathway has a capability of carrying out production of compound (I) from simple molecules such as compound (X), shown below, and acetaldehyde.
  • This approach is advantageous since this approach joins previously separate steps of production of compound (II), purification of compound (II), and oxidation of compound (II) to compound (I).
  • the cultivation of organisms carrying having said synthetic biological pathway is performed in one industrial fermentation process which immediately provides material capable of converting molecules such as compound (IX) and acetaldehyde into compound of formula (I).
  • Another embodiment of the present invention is obtaining the compound of formula (I), or salts, esters or stereoisomers thereof, in a one-pot process by reacting the starting materials for the DERA enzyme reaction in the presence of 2-deoxyribose-5-phosphate aldolase (DERA) enzyme and an enzyme capable of catalyzing oxidation or dehydrogenation, and optionally salifying, esterifying or stereoselectively resolving the product.
  • DERA 2-deoxyribose-5-phosphate aldolase
  • This embodiment contemplates to start from the compound of formula (X) in which R denotes R1-CH-R2 moiety of formula (I), and R1 and R2 being as defined hereinabove; and subjecting said compound (X) to reaction with acetaldehyde in the presence of the two enzymes, namely aldolase (DERA) enzyme and the enzyme capable of catalyzing oxidation or dehydrogenation.
  • R denotes R1-CH-R2 moiety of formula (I)
  • R1 and R2 being as defined hereinabove
  • This setup allows obtaining the compound of formula (I) in a single process step starting from relatively simple starting materials, e.g. acetaldehyde.
  • the reaction is industrially suitable, as it proceeds to completion within few hours. It renders intermediate purification steps superfluous.
  • the total amount of substrates added to the mixture is such that the total amount of the substrate (X) added would be from about 20 mmol per liter of the reaction mixture to about 2 mol per liter of the reaction mixture, in particular from about 100 mmol per liter of the reaction mixture to about 1.5 mmol per liter of the reaction mixture, more particular from about 200 mmol per liter of the reaction mixture to about 700 mmol per liter of the reaction mixture.
  • Acetaldehyde may be added by several means. In one aspect, acetaldehyde is added to the reaction mixture in one batch or more batches or alternatively continuously.
  • Acetaldehyde may be premixed with the substrate of formula (X) and added to the reaction mixture.
  • the total amount of acetaldehyde added to the reaction mixture is from about 0.1 to about 4 molar equivalents to the total amount of the acceptor substrate, in particular from about 2 to about 2.5 molar equivalents.
  • the pH-value used for the reaction is from about 4 to about11.
  • the pH used for reaction is from about 5 to about 10.
  • the pH-value used for reaction is from about 5 to about 8.
  • the pH-value will be maintained by a suitable buffer in a range from 52 to 7.5.
  • the pH-value as stated above may be controlled by, but not limited to, controlled addition of acid or base according to need as will be obvious to the person skilled in the art.
  • the pH used for the reaction described by the present invention may be optimized so that the compromise between optimal enzyme activity and optimal substrate and/or product stability is taken. It is understood herein that optimal enzymatic activity for different enzymes described in this invention may not be identical to optimal conditions for substate/product stability. A person skilled in art may find it benneficial to sacrifice some enzyme activity by adjusting conditions to suite substrate and/or product stability (or vice versa) to obtain optimal product yields.
  • aldolase enzyme optionally at least in part together with the enzyme capable of catalyzing oxidation or dehydrogenation are prepared in an aqueous solution (particularly each in a concentration 0.1 g/L to 3 g/L), optionally in the presence of a salt (in particular NaCl in a concentration from 50 to 500 mM and CaCl 2 or alternative Calcium salt in a concentration from 0,01 to 10mM).
  • a salt in particular NaCl in a concentration from 50 to 500 mM and CaCl 2 or alternative Calcium salt in a concentration from 0,01 to 10mM.
  • the aqueous solution may contain organic solvents miscible with water (in particular dimethyl sulfoxide in a concentration from 2 to 15 % V/V), and may be buffered to pH 4 to 11.
  • buffers can lower the yield of the aforementioned reaction that starts from the acetaldehyde by limiting the availability of aldolase-condensation intermediates, particularly first condensation reaction products as they may undergo a chemical reaction with a buffer.
  • aldolase-condensation intermediates particularly first condensation reaction products as they may undergo a chemical reaction with a buffer.
  • bis-tris propane reacts with said intermediates ((S)-3-hydroxy-4,4-dimethoxybutanal) giving ( S,Z )-2-(3-((1,3-dihydroxy-2-hydroxymethyl)propan-2-yl)(3-hydroxy-4,4-dimethoxybut-1-enyl)amino)propyl-amino)-2-(hydroxymethyl)propane-1,3-diol.
  • buffers that may react similarly are bis-tris, tricin, tris, bicin or any other buffer having a primary, secondary or tertiary amino group.
  • suitable buffers for adjusting pH, if this adjustment is needed are made with acids, bases, salts or mixtures thereof, in particular phosphoric acid and sodium hydroxide.
  • the buffer is a phosphate buffer.
  • phosphate buffer in a concentration 10 to 500 mM can be used.
  • the aqueous solution can also be prepared by adding the aldolase enzyme, optionally at least in part together with the enzyme capable of catalyzing oxidation or dehydrogenation to water and maintaining the pH-value during the reaction by means of an automated addition of inorganic acids, bases, salts or mixtures thereof.
  • the temperature used for the reaction starting from acetaldehyde is from about 10 to about 70 °C. In one embodiment, the temperature used for the reaction is from about 20 to about 50 °C. In another embodiment, the temperature used for the reaction is from about 25 to about 40 °C.
  • the temperature used for the reaction described by this invention may be optimized so that the compromise between optimal enzyme activity and optimal substrate and/or product stability is taken. It is understood herein that optimal enzymatic activity for different enzymes described in this invention may not be identical to optimal conditions for substate/product stability. A person skilled in art may find it beneficial to sacrifice some enzyme activity by adjusting conditions to suite substrate and/or product stability (or vice versa) to obtain optimal product yields.
  • either enzyme can be removed from the reaction mixture, for example by the addition of at least about 1 vol. of acetonitrile to 1 vol. of reaction mixture.
  • the enzyme can be removed by any salting out method known in the art.
  • the salting out is performed with the addition of ammonium sulfate of at least 5 % m/V.
  • the enzyme may be removed by filtrating or centrifuging the reaction mixture.
  • the pH of water solution of the product Prior the liquid/liquid extraction with chosen organic solvent the pH of water solution of the product may be adjusted to values between 1 and 12, preferably between 2 and 8, more preferably between 3 and 5. Drying of water residues in organic phase after extraction completion may be performed with but is not limited to adding salts listed: sodium sulfate, magnesium sulfate (monohydrate), calcium sulfate, calcium chloride, copper sulfate.
  • the aldolase enzyme and/or enzyme capable of catalyzing oxidation or dehydrogenation used can be prepared by any means known in the art, for example by methods of protein expression described in Sambrook et al. (1989) Molecular cloning: A laboratory Manual 2nd Edition, New York: Cold Spring Harbor Laboratory Press, Cold Spring Harb or.
  • Gene coding aldolase enzyme and/or enzyme capable of catalyzing oxidation or dehydrogenation can be cloned into an expression vector and the enzyme be expressed in a suitable expression host.
  • Modified versions of known aldolase enzyme or enzyme capable of catalyzing oxidation or dehydrogenation may be necessary or may result depending on cloning conditions and are encompassed in the present invention.
  • One aspect of present invention provides a process of oxidation or dehydrogenation of compound (II) or other compounds recited herein using a microorganism in any form described herein having enzyme capable of catalyzing oxidation or dehydrogenation reaction natively expressed.
  • a microorganism in any form described herein having enzyme capable of catalyzing oxidation or dehydrogenation reaction natively expressed.
  • such organism when cultivated and used as catalyst can convert compound (II) to corresponding lactone without the need for additional genetic modification of said microorganisms.
  • a methods of identifying such organisms is exemplified in hereby invention.
  • Non-limiting examples of such organism can be selected from vide diversity of bacteria, more particularly Escherichia, Corynebacterium, Pseudomonas, Streptomyces, Rhodococcus, Bacillus, Lactobacillus, Klebsiella, Enteorobacter, Acinetobacter, Rhizobioum. Methylobacterium, Kluyvera, Gluconobacter, Erwinia, Rahnella and Deinococcus.
  • a specially adapted expression system capable of translating 2-deoxyribose-5-phosphate aldolase (DERA) enzyme and an enzyme capable of catalyzing oxidation or dehydrogenation, and overexpressing both of the genes needed for said translation.
  • the term "overexpressing” as used herein refers to the expression under control of a strong promoter, or wherein the gene is expressed at high levels (compared to w.t. expression control) and is accumulated intracellularly or extracellularly.
  • the process of obtaining such a modified expression is known to a person skilled in the art. For example, cloning methods described in Sambrook et al.
  • the expression system comprises separate cells, wherein first cell overexpresses the gene for aldolase enzyme and second cell overexpresses the gene for the enzyme capable of catalyzing oxidation or dehydrogenation.
  • first cell overexpresses the gene for aldolase enzyme
  • second cell overexpresses the gene for the enzyme capable of catalyzing oxidation or dehydrogenation.
  • the cell system would be prokaryotic or eukaryotic.
  • the enzyme can be prepared synthetically.
  • the cell for preparing or hosting either of the enzymes can be a bacteria, yeast, insect cell or a mammalian cell.
  • the cell is bacteria or yeast and more preferably is bacteria, because bacteria or yeast cell are easier cultivated and grown.
  • the bacteria can be selected from the group of genera consisting of Escherichia, Corynebacterium, Pseudomonas, Streptomyces, Rhodococcus, Bacillus and Lactobacillus, preferably from Escherichia and Lactobacillus, more preferably Escherichia, particularly is Escherichia Coli.
  • the cell can be selected from the group of genera consisting of Saccharomyces, Pichia, Shizosaccharomyces and Candida, preferably Saccharomyces.
  • the examples of mammalian cells are Chinese hamster ovary cell or a hepatic cell, preferably is Chinese hamster ovary cell.
  • Another embodiment of the invention is a process for the preparation of compound (I), in particular an industrial fermentative process, wherein the process comprises the step of cultivation of a microorganism capable of oxidation of compound (II), wherein the said microorganism is brought in contact with compound (II).
  • Yet another embodiment of this invention is a process for the preparation of compound(I), in particular an industrial fermentative process, wherein the process comprises the step of cultivation of a microorganism, capable of oxidation of compound (II), wherein said microorganism is brought in contact with another microorganism having ability of enzymatic production of (II), particularly by catalysis of DERA and wherein substrates which allow production of compound (II) are provided to the reaction mixture.
  • a preferred embodiment of his invention is a process for the preparation of compound (I), in particular an industrial fermentative process, wherein the process comprises the step of cultivation of a microorganism, capable both of production as well as oxidation/dehydrogenation of compound (II) and wherein substrates which allow production of compound (II) are provided to the reaction mixture.
  • the process according to the present invention comprises the following steps:
  • a microorganism is identified and/or genetically modified in order to obtain both properties described in step a1) and step a2).
  • the invention specifically relates to a genetically modified strain of a microorganism wherein the genetic material of the strain comprises at least one over expressed gene coding for and enzyme capable of catalysing aldol condensation to form compound (II), more specifically a gene encoding DERA enzyme.
  • step a1) and step a2) Procedures to identify and/or generate genetically modified microorganisms as described in step a1) and step a2) are exemplified in detail in this invention, however a skilled person will immediately find alternative procedures which may lead to the same desired properties of said microorganisms.
  • cloning methods described in Sambrook et al. (1989) Molecular cloning: A laboratory Manual 2nd Edition, New York: Cold Spring Harbor Laboratory Press, Cold Spring Harb or, can be used.
  • the genes for the enzymes can be for example cloned on the same or different vector and transformed into a cell.
  • the expression system comprises separate cells, wherein first cell overexpresses the gene for aldolase enzyme and second cell overexpresses the gene for the enzyme capable of catalyzing oxidation or dehydrogenation.
  • first cell overexpresses the gene for aldolase enzyme
  • second cell overexpresses the gene for the enzyme capable of catalyzing oxidation or dehydrogenation.
  • the cell system would be prokaryotic or eukaryotic.
  • the enzyme can be prepared synthetically.
  • the cell for preparing or hosting either of the enzymes can be a bacteria, yeast, insect cell or a mammalian cell.
  • the cell is bacteria or yeast and more preferably is bacteria, because bacteria or yeast cell are easier cultivated and grown.
  • the bacteria can be selected from the group of genera consisting of Escherichia, Corynebacterium, Pseudomonas, Streptomyces, Rhodococcus, Bacillus, Lactobacillus, Klebsiella, Enteorobacter, Acinetobacter, Rhizobioum. Methylobacterium, Kluyvera, Gluconobacter, Erwinia, Rahnella and Deinococcus.
  • the microorganisms may be selected from Klebsiella pneumoniae, Acinetobacter calcoaceticus, Methylobacterium extorquens, Kluyvera intermedia, Enterobacter, Gluconobacter oxydans, Pseudomonas aeruginosa, Erwinia amylovora, Rahnella aquatilis, Deinococcus radiodurans, Corynebacterium glutamicum, Escherichia coli, Bacillus licheniformis, Lactobacillus lactis, most preferably from: Escherichia coli, Gluconobacter oxydans, Acinetobacter calcoaceticus and Kluyvera intermedium.
  • yeast the cell can be selected from the group of genera consisting of Saccharomyces, Pichia, Shizosaccharomyces and Candida , preferably Saccharomyces and Pichia.
  • a microorganism is identified and/or genetically modified in order to obtain both properties described in step a1) and step a2).
  • the invention specifically relates to a genetically modified strain of a microorganism wherein the genetic material of the strain comprises at least one over expressed gene coding for and enzyme capable of catalysing aldol condensation to form compound (II), more specifically a gene encoding DERA aldolase enzyme.
  • the present invention provides an exemplified method of constructing synthetic biological pathway, examplified with E. coli as a host microorganism, wherein DERA (deoxyribose 5-phosphate aldolase), PQQ dependant dehydrogenase and PQQ biosynthetic pathway genes are expressed simultaniously.
  • DERA deoxyribose 5-phosphate aldolase
  • PQQ dependant dehydrogenase and PQQ biosynthetic pathway genes are expressed simultaniously.
  • Providing the respiratory chain of the host organism said synthetic biological pathway has a capability of carrying out production of compound (I) from simple molecules such as compound (X) and acetaldehyde.
  • One aspect described in this invention is to cultivate microorganisms described in step a1 and a2 simultaneously or independently.
  • Cultivation of the microorganisms as described in the present invention can be carried out by methods known to a person skilled in art. Cultivation processes of various microorganisms are for example described in the handbook " Difco & BBL Manual, Manual of Microbiological Culture Media” (Zimbro M.J. et al., 2009, 2nd Edition. ISBN 0-9727207-1-5 ).
  • the production of seed microorganism which can be used in the main fermentation process for the production of (I) starts from a colony of said microorganism.
  • the process according to the present application comprises the preparation of frozen stock of described microorganism. This preparation of frozen stock may be carried out using method known in the state of art, such as using a liquid propagation medium.
  • this frozen stock of microorganism is used to produce a vegetative seed medium by inoculation to a vegetative medium.
  • the seed medium may be transferred aseptically to a bioreactor.
  • the cultivating of seed microorganism can be carried out under the conditions (e.g. pH and temperature) as in the main fermentation process (described under step c).
  • the main fermentation process using a microorganism as described in the present application is carried out in a bioreactor in particular under agitation and/or aeration.
  • cultivation of a microorganism used for the process for the production of compound (I) as described in the present application is carried out under submerged aerobic conditions in aqueous nutrient medium (production medium), containing sources of assimilable carbon, nitrogen, phosphate and minerals. Additional compounds may be added to the production medium during or after the cultivation process in order to obtain appropriate enzymatic activities. These may include expression inducers , sources of cofactors and or compounds allowing maintanence of genetic elements (such as antibiotics).
  • the main fermentation process comprises the inoculation of production medium with seed microorganism obtained in step b) in particular by asepticall transfer into the reactor. It is preferred to employ the vegetative form of the microorganism for inoculation.
  • the addition of nutrient medium (production medium) in the main fermentation process into the reactor can be carried out once or more batch-wise or in a continuous way. Addition of nutrient medium (production medium) can be carried out before and/or during the fermentation process.
  • the preferred sources of carbon in the nutrient media can selected from dextrin, glucose, soluble starch, glycerol, lactic acid, maltose, fructose, molasses and sucrose as exemplified below.
  • the preferred sources of nitrogen in the nutrient media are ammonia solution, yeast extract, soy peptone, soybean meal, bacterial peptone, casein hydrolysate, L-lysine, ammonium sulphate, corn steep liquor and other.
  • Inorganic/mineral salts such as calcium carbonate, sodium chloride, sodium or potassium phosphate, magnesium, manganese, zinc, iron and other salts may also be added to the medium.
  • anti-foaming agents such as silicone oil, fatty oil, plant oil and the like.
  • a silicone-based anti-foaming agent may be added during the fermentation process to prevent excessively foaming of the culture medium.
  • Expression inducers such as isopropyl ⁇ -D-1-tiogalaktopyranoside (IPTG), Arabinose, Tetracycline, indoleacrilate etc. may be added to the culture medium.
  • cofactors such as PQQ (pyrroloquinoline quinone), NAD(P), FAD may be added to improved the activity of involved enzymes.
  • IPTG and PQQ may be used.
  • the fermentative process could be performed in aerobic conditions with agitation and aeration of production medium. Agitation and aeration of the culture mixture may be accomplished in a variety of ways.
  • the agitation of production medium may be provided by a propeller or similar mechanical device and varied to various extents according to fermentation conditions and scale.
  • the aeration rate can be varied in the range of 0.5 to 2.5 WM (gas volume flow per unit of liquid volume per minute (volume per volume per minute)) with respect to the working volume of the bioreactor.
  • the main fermentation process by the present process is carried out at a pH in the range of about 6.3 to 8.5 and temperature in the range of 18 to 37 °C.
  • the pH is in the range of about 6.5 to 8.3 and the temperature is in the range of about 21 to 31 °C.
  • the cultures are incubated for 16 to about 300 hours, more preferably for about 30 to 70 hours.
  • Another embodiment of in this invention encompasses cultivation of microorganisms described in step a) simulaniously or independatly. Likewise it is also possible to conduct reactions for preparation of compound (II) and compound (I) separately or simultanuosly, successively or in a one pot manner.
  • dehydrogenase/oxidase enzyme is prepared after step c) in an aqueous solution (particularly in a concentration range from 0.1 g/L to 300 g/L) in present of salt (in particular NaCl in concentration range from 50 to 500 mM), diluted or concentrated to said concentration range.
  • salt in particular NaCl in concentration range from 50 to 500 mM
  • aqueous solution may be buffered to pH 4.0 to 11, preferably to pH 5.0. to 10.0, more preferably to 5.0 to pH 8.0. Most preferably the solution is buffered to about pH5.2 to obout pH 7,5.
  • Suitable buffers can be prepared from: acids, bases, salts or mixtures thereof, and any other buffer system known in the art except those possessing primary, secondary or tertiary amino group.
  • phosphate buffer in concentration 10 to 500 mM may be used.
  • the aqueous solution can be prepared by adding the said dehydrogenase/oxidase enzyme to water and maintaing pH during the reaction by means of automated addition of inorganic or organic acids, bases, salts of mixtures thereof.
  • Compound (II) is added into the culture of microorganism having compound (II) oxidation/dehydrogenation capability.
  • PQQ may be added in concentration from about 0.1nM to about 5mM. In particular from about 1nM to about 100uM of PQQ can be provided. More preferably the PQQ is provided in concentration from 100nM to about 2 ⁇ M. Most preferably the PQQ is provided in the minimal amount which allows maximal activity of the said catalyst. Practically this is in the range of 250nM to 1 ⁇ M final concentration of PQQ.
  • an artificial electron acceptor is added in equimolar concentration to compound II as described in this invention.
  • the process is preformed by using microorganisms capability of accepting electrons formed during dehydrogenation / oxidation reaction into its intrinsic respiratory chain.
  • Compound (II) may be provided as partiallly or fully isolated compound (II) obtained from previous reaction mixture containing DERA aldolase under aldolase-catalysed aldol condensation conditions or from other sources (organic synthesis).
  • Compound (II) may be added in by any means and rates as described within this invention in a final concentration from about 20mM to about 1 M, preferably from about 50mM to about 700mM, most preferred concentrations are between 100mM and 500mM.
  • step c additional nutrients in order to support microorganisms viability may be added in similar way as described in step c). Practically it may be beneficial to provide additional carbon source to said reaction mixture as described within hereby invention, optionally independently or in addition also a nitrogen source.
  • General reaction conditions which allow dehydrogenation/ oxidation of compound (II) in this specific aspect are defined as 'aldose dehydrogenation/oxidation conditions' as provided by this invention.
  • the compound (II) as substrate for oxidase/dehydrogenase to obtain compound (II) may be added to the reaction mixture in one batch or more batches.
  • the total amount of substrate added to the mixture is such that the total amount of compound (II) added would be from about 20 mmol per liter of reaction mixture to about 1.5 mol per liter of reaction mixture, more particular from about 100 mmol per liter of reaction mixture to about 700 mmol per liter of reaction mixture.
  • the substrates are added continuously to the reaction mixture by means of programmable pump at specific flow rate at any given time of the reaction. Optimally, the flow rate is determined as maximum flow rate where the substrate is not accumulating in the reaction mixture. In particular this allows minimal concentrations of undesired products. In another embodiment the inhibitory effect of substrate can be further minimized using correct addition strategy.
  • the invention provides a variant described as d1), however in this case the compound (II) is provided in situ directly in form of reaction mixture obtained by reacting DERA aldolase with compound (X) and acetaldehyde by methods known in the art.
  • this approach uses the advantage of performing a one-pot reaction and thus significantly impacting the simplicity of the combined process. It is advantageous to use whole cell catalyst containing DERA aldolase in the proceeding step in accordance to 'aldolase-catalysed aldol condensation conditions"
  • Both catalysts are obtained by fermentation process simultaneously or independently (as described in step c) and catalysts are then transferred into a suitable vessel or reactor, preferably joined or left in same fermenter used for obtaining the catalyst.
  • Another yet similar aspect of the process both enzymatic activities are present in a single microorganism which is preferably obtained as described in step c.
  • a simultaneous process containing using DERA aldolase and enzyme capable of oxidation/dehydrogenation and thus providing compound (I).
  • the acetyloxyacetaldehyde (CH 3 CO 2 CH 2 CHO) as substrate for DERA aldolase to obtain compound (II) may be added to the reaction mixture continuously or alternatively the acetyloxyacetaldehyde (CH 3 CO 2 CH 2 CHO) is added to the reaction mixture in one batch or more batches.
  • the total amount of substrates added to the mixture is such that the total amount of acetyloxyacetaldehyde (CH 3 CO 2 CH 2 CHO) added would be from about 20 mmol per liter of reaction mixture to about 1.5 mol per liter of reaction mixture, more particular from about 100 mmol per liter of reaction mixture to about 700 mmol per liter of reaction mixture.
  • Acetaldehyde may be added by several means.
  • the acetaldehyde is added to the reaction mixture in one batch or more batches or alternatively continuously.
  • Acetaldehyde may be premixed with acetyloxyacetaldehyde (CH 3 CO 2 CH 2 CHO) and added to the reaction mixture.
  • the total amount of acetaldehyde added to the reaction mixture is from about 0.1 to about 4 molar equivalents to total amount of acceptor substrate acetyloxyacetaldehyde (CH 3 CO 2 CH 2 CHO), in particular from about 1 to about 3 molar equivalents, more preferably from about 2 to 2.5 molar equivalents. In particular, this allows minimal concentrations of undesired products.
  • PQQ is added into the reaction mixture in concentrations from about 0.05 ⁇ M to about 10 mM, more preferably 0.1 uM to about 1 mM.
  • the substrates are added continuously to the reaction mixture by means of programmable pump at specific flow rate at any given time of the reaction. The flow rate is determined as maximum flow rate where the substrates are not accumulating in the reaction mixture. In particular this allows minimal concentrations of undesired products.
  • the inhibitory effect of substrates can be further minimized using correct addition strategy.
  • the temperature used for dehydrogenase/oxidase-catalysed reaction is from about 10 °C to about 70 °C.
  • the temperature used for dehydrogenase/oxidase reaction is from about 20 to about 50 °C. In one embodiment the temperature used for dehydrogenase/oxidase reaction is from about 25 °C to about 40 °C.
  • the reaction is industrially suitable, as it proceeds to completion within few hours.
  • aldose dehydrogenation/oxidation conditions refers to any dehydrogenation/oxidation conditions known in the art that can be catalysed by any dehydogenase/oxidase enzyme, as described herein.
  • the dehydrogenase/oxidase activity conditions are such that allow forming and accumulation of desired product.
  • these conditions include in one aspect that the dehydrogenase/oxidase is an active enzyme provided at sufficient load to be able to perform the dehydrogenation/oxidation.
  • the compound (II) as substrate is present in the reaction mixture in an amount that displays minimal inhibition of the activity of the aldolase.
  • an dehydrogenase/oxidase for use in the present invention may be any enzyme that has dehydrogenase/oxidase activity towards compound of formula (II).
  • the dehydrogenase/oxidase enzymes include but are not limited to: GDH 01, GDH 02, GDH 03, GDH 04, GDH 05, GDH 06, GDH 07, GDH 08, GDH 09, GDH 10, GDH 11, GDH 12, GDH 13, GDH 14, GDH 15, GDH 16, GDH 17, GDH 18, GDH 19, GDH 20, GDH 21, GDH 22, GDH 23, GDH 24 and GDH 25, wherein each enzyme is identified by it's corresponding nucleotide sequence or respective codon optimized nucleotide sequence or aminoacid sequence as set forth in sequence listing above.
  • the dehydrogenase/oxidase catalyst described herein can be used in any biologically active form provided in this invention.
  • catalyst able to obtain compound (I) will be provided in a suitable vessel or reactor, and the compound (II) will be added batch-wise or continuously, or provided by, at least in part simultaneous production of compound (II), preferably in same vessel under "aldolase-catalysed aldol condensation conditions".
  • aldolase-catalysed aldol condensation conditions refers to any aldol condensation conditions known in the art that can be catalysed by any aldolase, as described for example in WO2008/119810 A2 .
  • the aldolase-catalysed aldol condensation conditions are such that allow forming and accumulation of desired product, more preferably that the substituted acetaldehyde R 1 CO 2 CH 2 CHO, more particularly acetyloxyacetaldehyde (CH 3 CO 2 CH 2 CHO) as substrate and acetaldehyde are present in the reaction mixture in an amount that displays minimal inhibition of the activity of the aldolase, in another aspect that the temperature, pH, solvent composition, agitation and length of reaction allow accumulation of desired product and thus forming corresponding lactole (compound II).
  • the DERA aldolase described therein can be used in any biologically active form provided in said invention.
  • the substrates for DERA aldolase, the compounds of formula (X) are selected according to the corresponding compound (II). These products having a masked aldehyde group are key intermediates in WO2008/119810 A2 and WO2009/092702 A2 allowing further steps in preparation of statins, in particular, substrates yielding a product with aldehyde group are preferred.
  • the compound (X) may be in particular acetyloxyacetaldehyde (CH 3 CO 2 CH 2 CHO).
  • Isolation and/or purification of compound (I), which was produced in the main fermentation process from the said medium, may be carried out in a further separation step.
  • whole cell catalyst present in reaction mixture may be removed by any known type of filtration/flocculation/sedimentation/centrifugation procedure or further steps of the isolation are performed on whole cell containing reaction mixture.
  • the isolation of compound (I) may be carried out by adsorbtion to an adsorbent capable of binding compound (I) at significant levels and releasing compound (I) upon elution conditions such as replacement of the medium by a more nonpolar compound.
  • Adsorbent may be selected from but not limited to: silica gel, zeolites, activated carbon, AmberliteTM XADTM adsorbent resins, AmberliteTM and AmberliteTM FP ion exchange resins etc.
  • liquid-liquid extraction may be carried out using water miscible solvents such as acetonitrile or methanol and supplementing the mixture with high concentration of salts, optionaly Sodium chloride in a so colled “salting out” extraction procedure. In this process separation of phases is observed and the compound (I) is preferentialy distributed in solvent rich phase.
  • water miscible solvents such as acetonitrile or methanol
  • the extraction solvent for the liquid/liquid extraction is chosen from any a number of water immiscible or poorly miscible solvents.
  • the solvent may be selected from but is not limited to: methylene chloride, diethyl ether, propionyl acetate, methyl t-butyl ether (MTBE), nitromethane, pentane, hexane, heptane, 1,2-dichloroethane, chloroform, carbon tetrachloride, n-butanol, n-pentanol, benzene, toluene, o-, m-, p-xylene, cyclohexane, petroleum ether, triethylamine.
  • the pH of water solution of the product Prior the liquid/liquid extraction with chosen organic solvent the pH of water solution of the product may be adjusted to values between 1 and 9, preferably between 2 and 8, more preferably between 2 and 5. Drying of water residues in organic phase after extraction completion may be performed with but is not limited to adding salts listed: sodium sulfate, magnesium sulfate (monohydrate), calcium sulfate, calcium chloride, copper sulfate.
  • the pH of the reaction mixture is first corrected to value from about 1 to 9, preferably from about 2 to 8, more preferably from about 2 to 5. Most preferably pH is corrected to about 3,5 using and alkaline compound such as NaOH, NaH 2 CO 2 etc.
  • Ethyl acetate is added at least 1 time by the addition of at least 1 volume of ethyl acetate to 1 volume of reaction mixture, preferably at lest 3 times by the addition of 1 volume of ethyl acetate to 1 volume of reaction mixture.
  • the steps of adding ethyl acetate and separationg the extract are carried out until no more than 5% of compound (I) is present in aqueous phase.
  • Ethyl acetate fractions are collected, dried with any of the drying salts known in the art, preferably CaCl 2 or MgSO 4 or other methods of water stripping known in the art.
  • the obtained ethylacetate extract can be evaporated to yield isolated compound (I) in a form of yellow - amber oul at room temperature or can alternatively proceede into further steps in order tu yield pharmaceutically usefull compounds, preferably statins.
  • the compound of formula (I) can be further transformed to an API, preferably statin, or a pharmaceutically acceptable salt thereof, by subjecting said compound (I) to conditions sufficient to prepare the API, preferably statin.
  • a statin or salt, ester or stereoisomer thereof is prepared by (i) bringing in contact the compound of formula (II) as defined hereinabove with an enzyme capable of catalyzing oxidation or dehydrogenation, to prepare a compound of formula (I) as defined hereinabove, (ii) subjecting said compound (I) to conditions sufficient to prepare a statin; and (iii) optionally salifying, esterifying or stereoselectively resolving the product.
  • the compound of formula (II) can be prepared by using 2-deoxyribose-5-phosphate aldolase (DERA, EC 4.1.2.4) enzyme.
  • the reaction setup can be arranged to introduce both enzymes substantially simultaneously or subsequently, at once or continuously, in one batch or in intermittent batches.
  • the compounds of formula (II) and (I) are prepared at least in part simultaneously, more preferably substantially simultaneously. It is advantageous to use enzymes for preparing compounds of formula (I) and/or (II) in the case of enzymes, because the product immediately contains the correct spacious orientation of the substituents and no further purification or separation is needed. In the event that other stereoisomers are needed, the process can be combined with methods of stereospecific chemistry known to the skilled person.
  • the statin prepared is lovastatin, pravastatin, simvastatin, atorvastatin, cerivastatin, rosuvastatin, fluvastatin, pitavastatin, bervastatin, or dalvastatin, more preferably atorvastatin, rosuvastatin or pitavastatin, particularly is rosuvastatin.
  • condition sufficient to produce an API, preferably statin refers to those means described in the art, including those means described herein for conversion of the compound of formula (I) further to the API, preferably statin.
  • statin is prepared, the skilled person would choose the chemical route by selecting the proper R1 and R2, or R.
  • R1, R2 and R are chosen to represent the statin skeleton, the compound of formula (I) is already a statin molecule ro an "advanced" intermediate thereof. Nevertheless, modifying the statin like for example by opening the lactone ring, forming the salt or the ester or resolving desired stereoisomers from the mixture of stereoisomers is also possible.
  • statin is to be prepared by first providing a lactone and then coupling it to the statin skeleton, a reaction scheme 2 can be followed.
  • the compound of formula (I) its hydroxyl group in position 4 can be protected with the protecting group P (formula (XI)), which can be any conventionally used protecting group, in particular is silyl protecting group.
  • the compound can be brought in the form of an aldehyde or its hydrate (formulas (XII) and (XIII), respectively).
  • Compound of formula (XII), or hydrate thereof (XIII), obtained from I can be further used to prepare statin by reacting the compound of formula (XII), or hydrate thereof (XIII), under the condition of a Wittig coupling with a heterocylic or alicyclic derivative (statin skeleton) followed by hydrogenation when needed.
  • (2 S ,4 R )-4-( P -oxy)-6-oxo-tetrahydro-2 H -pyran-2-carbaldehyde (XII) can be reacted under the conditions of a Wittig coupling (in the presence of a base) with a ((4-(4-fluorophenyl)-6-isopropyl-2-(N-methylmethylsulfonamido)pyrimidin-5-yl)methyl)triphenyl-phosphonium halide or any other ((4-(4-fluorophenyl)-6-isopropyl-2-( N -methylmethylsulfon-amido)pyrimidin-5-yl)methyl)phosphonium salt or alternatively di- i -propyl( ⁇ 4-(4-fluorophenyl)-6-isopropyl-2-[methyl(methylsulfonyl)amin
  • lithium hexamethyldisilazane LiHMDS
  • potassium hexamethyldisilazane KHMDS
  • sodium hexamethyldisilazane NaHMDS
  • lithium diisopropylamide LDA
  • sodium hydride butyllithium or Grignard reagents, preferably sodium hexamethyldisilazane
  • the source is the hydrate form XIII or a mixture of XII and the hydrate form XIII thereof, which is dissolved in ethers selected from THF, Et 2 O, i -Pr 2 O, t BuMeO; hydrocarbons selected from: pentane, hexane, cyclohexane, methylcyclohexane, heptane; aromatic hydrocarbons selected from toluene or the chlorinated derivatives thereof; chlorinated hydrocarbons selected from: chloroform and dichloromethane or in mixtures of those solvents, water released from the hydrate should be removed prior to the addition to the formed ylide solution.
  • ethers selected from THF, Et 2 O, i -Pr 2 O, t BuMeO
  • hydrocarbons selected from: pentane, hexane, cyclohexane, methylcyclohexane, heptane
  • aromatic hydrocarbons selected from toluene
  • the preferred solvents for the reaction are anhydrous toluene and dichloromethane.
  • the reaction can be performed at temperatures between -80 °C and 90 °C preferably at 0 to 90 °C, more preferably at 80 - 90 °C.
  • the reaction is accomplished in 1 - 12 hours.
  • Isolation of the crude product with extraction can be performed with AcOEt, ethers or alkanes, preferably with t BuMeO.
  • the protecting group may be removed and the lactone opened to produce a rosuvastatin free acid or a salt thereof, optionally an amine, which may be converted to hemicalcium salt.
  • the deprotection can be performed at temperatures between 0 °C to 80 °C.
  • a suitable solvent preferably a solvent selected from alcohols, acetic acid, THF, acetonitrile, methyltetrahydrofuran, dioxane, CH 2 Cl 2 , more preferably in alcohols and a mixture of THF/AcOH.
  • the usual deprotecting reagents may be used, such as tetra- n -butylammonium fluoride, ammonium fluoride, AcCl, FeCl 3 , TMSCl/HF ⁇ 2H 2 O, chloroethylchloroformate (CEC), Ph 3 PCH 2 COMeBr.
  • the opening of the lactone preferably takes place in a 4:1 to 2:1 mixture of THF/H 2 O as well as in pure THF at temperatures between 20 °C to 60 °C with a suitable alkali such as NaOH, KOH, ammonia or amines.
  • the hydrolysis is accomplished in 30 minutes (at 60 °C) to 2 hours (at 20 °C).
  • evaporation of THF can be conducted at temperatures between 10 °C to 50 °C under the reduced pressure, and conversion to the calcium salt, preferably by the addition of Ca(OAc) 2 ⁇ ⁇ H 2 O, which can be added in one portion or dropwise in 5 to 60 minutes, can be performed at temperatures between 0 °C to 40 °C.
  • the resulting suspension can be stirred at temperatures between 0 °C to 40 °C from 30 minutes to 2 hours.
  • the details of such reaction are known in the art, including in, but not limited to, WO2008/119810 .
  • the API preferably statin, obtained by any of the aforementioned embodiments, can be formulated in a pharmaceutical formulation.
  • the methods for preparing a pharmaceutical formulation with the API, preferably statin are known to the person skilled in the art. Generally, one can chose among preparing formulations such as powder, granulate, tablet, capsule, suppository, solution, ointment, suspension, foam, patch, infusion, solution for injection, or the like.
  • the formulation can be changed in order to modify specific aspects of the API like for example release, stability, efficacy or safety.
  • the skilled person knows how to select proper administration route for said API. Based on that, he can choose proper formulation.
  • excipients needed for formulating the pharmaceutical formulation.
  • the skilled person can select from excipients and additives to formulate the pharmaceutical formulation.
  • Suitable excipients may be, for example, binder, diluent, lubricant, disintegrant, filler, glidant, solvent, pH modifying agent, ionic strength modifying agent, surfactant, buffer agent, anti-oxidant, colorant, stabilizer, plasticizer, emulsifier, preservatives, viscosity-modifying agent, passifier, flavouring agent, without being limited thereto, which can be used alone or in combination.
  • the method can involve, depending on the selected formulation, mixing, grinding, wet granulation, dry granulation, tabletting, dissolving, lyophilisation, filling into capsules, without being limited thereto.
  • an enzyme capable of catalyzing oxidation or dehydrogenation can be generally used for preparing an API or intermediate thereof being compatible with the enzymatic system(s) disclosed herein.
  • This aspect of the invention preferably relates to synthetic API or intermediate thereof respectively categorisable as substituted or unsubstituted dideoxyaldose sugars, lactols (optionally containing multiple hydroxyl groups) and synthetic non-natural alcohols as possible substrates, and (optionally further hydroxylated) lactons or esters as possible products.
  • synthetic API or intermediate thereof respectively categorisable as substituted or unsubstituted dideoxyaldose sugars, lactols (optionally containing multiple hydroxyl groups) and synthetic non-natural alcohols as possible substrates, and (optionally further hydroxylated) lactons or esters as possible products.
  • the substrates or products of such pathways in the nature include methanol, ethanol, formaldehyde, acetaldehyde, methanoic, acetic acid, monosaccharide, disaccharide, trisaccharide, glucuronic acid, especially methanol, ethanol, formaldehyde, acetaldehyde, methanoic, acetic acid, monosaccharide, glucuronic acid, and particularly ethanol, acetic acid and monosaccharide.
  • the enzyme capable of catalyzing oxidation or dehydrogenation is used for preparing the compound of formula (I), wherein the formula (I) is as defined hereinabove.
  • the enzyme capable of catalyzing oxidation or dehydrogenation is used for preparing the compound of formula (I), wherein the formula (I) is as defined hereinabove.
  • it is the most efficient to use the enzyme to act upon the compound of formula (II), wherein the formula (II) is as defined hereinabove.
  • Specific alternatives of the use will be immediately apparent to the skilled person when other embodiments, aspects, or preferred features of the invention disclosed hereinabove are taken into account.
  • a possible useful further definition of the substrate and thus starting compound useful for the preparation of appropriate APIs or intermediates thereof is given by the following formula XIV, hence leading to a corresponding product compound defined by formula XV.
  • Q is any desired structural moiety, for example selected from the groups of R1, R2 and R5 defined hereinabove, optionally with an intermediate linker molecule between R1, R2 or R5 and the lactol/lactone ring.
  • the non-lactol hydroxyl group not being oxidyzed can be positioned at any position of the lactol/lactone ring.
  • the enzyme capable of catalyzing oxidation or dehydrogenation is used for preparing an API or intermediate thereof simultaneously or subsequently with a DERA enzyme.
  • aldose dehydrogenases have been prepared as descried in the following:
  • the genomic DNA from E. coli DH5 ⁇ was isolated using Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to manufacturer's instructions. Isolation of genomic DNA was made using overnight culture of E. coli grown on LB medium at 37 °C. Amplification of gene ylil was performed by PCR using oligonucleotide primers GCGCCATATGCATCGACAATCCTTT (SEQ ID NO. 71) and GCGCGCTCAGGCTAATTGCGTGGGCTAACTTTAAG (SEQ ID NO. 72).
  • Amplification was performed by Pfx 50 DNA polymerase (Invitrogen, Calsbad, CA, USA) as follows: an initial denaturation at 94 °C for 10 min, followed by 30 cycles of 45 s at 94 °C, 45 s at 52 °C, and 150 s at 68 °C. Final elongation was performed 420 s at 68 °C.
  • a 1.2-kb DNA fragment containing ylil (SEQ ID NO. 01) was separated by agarose gel electrophoresis and purified. The product was ligated into plasmid pGEM T-Easy (Promega, Madison, WI, USA) in a T4 ligase reaction.
  • the plasmid construct was cleaved with restriction endonucleases Nde I and Sal I, the resulting fragments were separated on agarose gel electrophoresis and 1.2 kb fragment containing ylil was purified.
  • An expression vector pET30a(+) (Novagen Inc., Madison, WI, USA) was cleaved using the same aforementioned restriction endonucleases and purified.
  • the 1.2 kb fragment containing ylil gene was assembled with the cleaved expression vector in a T4 ligase reaction. E. coli JM109 cells were transformed with the obtained ligation reaction and kanamycin resistant colonies were cultured. Afterwards plasmid DNA was isolated.
  • the resulting construct was designated pET30/Ylil and sequenced for confirmation of the gene sequence.
  • the cloning procedure was performed to allow expression of protein having sequence (SEQ ID NO. 02) including leader sequence for Ylil translocation to periplasm.
  • the aldose dehydrogenase expressing organism was prepared by transforming BL21 (DE3) competent cells with said plasmid.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the genomic DNA from E. coli DH5 ⁇ was isolated using Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to manufacturer's instructions. Isolation of genomic DNA was made using overnight culture of E. coli grown on LB medium at 37 °C. Amplification of gene ylil was performed by PCR using oligonucleotide primers GCGCCATATGGCTCCTGCAACGGTAAAT (SEQ ID NO. 73) and GCGCGCTCAGGCTAATTGCGTGGGCTAACTTTAAG (SEQ ID NO. 72).
  • Amplification was performed by Pfx 50 DNA polymerase (Invitrogen, Calsbad, CA, USA) as follows: 94 °C for 10 min, followed by 30 cycles of 45 s at 94 °C, 45 s at 50 °C, and 150 s at 68 °C. Final elongation was performed 420 s at 68 °C.
  • a 1.2-kb DNA fragment containing ylil (SEQ ID NO. 03) was separated by agarose gel electrophoresis and purified. The product was ligated into plasmid pGEM T-Easy (Promega, Madison, WI, USA) in a T4 ligase reaction.
  • the plasmid construct was cleaved with restriction endonucleases Nde I and Sal I, the resulting fragments were separated on agarose gel electrophoresis and 1.2 kb fragment containing ylil was purified.
  • An expression vector pET30a(+) (Novagen Inc., Madison, WI, USA) was cleaved using the same aforementioned restriction endonucleases and purified.
  • the 1.2 kb fragment containing yli l gene was assembled with the cleaved expression vector in a T4 ligase reaction.
  • E. coli JM1 09 cells were transformed with the obtained ligation reaction and kanamycin resistant colonies were cultured. Afterwards plasmid DNA was isolated.
  • the resulting construct was designated pET30/YliI_C and sequenced for confirmation of the gene sequence.
  • the cloning procedure was performed with a target to provide protein Ylil expressed in cytoplasm with coding sequence (SEQ ID NO. 04)
  • the aldose dehydrogenase expressing organism was prepared by transforming BL21 (DE3) competent cells with said plasmid.
  • the procedure of expression of E. coli Ylil_C was undertaken as described in Procedure 1A. After expression cells were harvested and whole cell catalysts were obtained as described in Procedure 1B to obtain living whole cell catalysts. To obtain resting whole cell catalysts Procedure 1C was undertaken.
  • a nucleotide sequence encoding a gene and leader sequence for transportation to periplasm (PQQ GdhB, A. calcoaceticus GeneBank #X15871) was optimized for expression in E. coli and DNA was chemically synthesized (Geneart, Regensburg, Germany) (SEQ ID. NO. 5).
  • E. coli JM109 cells were transformed with artificial plasmid bearing nucleotide sequence gdhB and kanamycin resistant colonies were cultured.
  • plasmid DNA was isolated and the construct was cleaved with restriction endonucleases Nde I and Hin dIII, the resulting fragments were separated on agarose gel electrophoresis and 1.5 kb fragment was purified (SEQ ID NO. 05).
  • An expression vector pET30a(+) (Novagen Inc., Madison, WI, USA) was cleaved using the same aforementioned restriction endonucleases and purified.
  • the 1.5 kb fragment containing gdhB gene was assembled in the cleaved expression vector in a T4 ligase reaction.
  • E . coli JM109 cells were transformed with the obtained ligation reaction and kanamycin resistant colonies were cultured.
  • plasmid DNA was isolated.
  • the resulting construct was designated pET30/GdhB and sequenced for confirmation of the gene sequence.
  • the cloning procedure was performed with a target to allow expression of protein having sequence SEQ ID NO. 06.
  • the aldose dehydrogenase from A. calcoaceticus expressing organism was prepared by transforming BL21 (DE3) competent cells with said plasmid.
  • the procedure of expression of E . coli GdhB was undertaken as described in Procedure 1A. After expression cells were harvested and whole cell catalyst was obtained as described in Procedure 1B to obtain living whole cell catalysts. To obtain resting whole cell catalyst Procedure 1C was undertaken.
  • E . coli JM109 cells were transformed with artificial plasmid bearing nucleotide sequence gdhB_therm and ampicillin resistant colonies were cultured. Afterwards plasmid DNA was isolated and the construct was cleaved with restriction endonucleases Nde I and Hin dIII, the resulting fragments were separated on agarose gel electrophoresis and 1.5 kb fragment was purified (SEQ ID NO. 07).
  • An expression vector pET30a(+) (Novagen Inc., Madison, WI, USA) was cleaved using the same aforementioned restriction endonucleases and purified.
  • the 1.5 kb fragment containing gdhB_therm gene was assembled in the cleaved expression vector in a T4 ligase reaction.
  • E . coli JM109 cells were transformed with the obtained ligation reaction and kanamycin resistant colonies were cultured. Afterwards plasmid DNA was isolated. The resulting construct was designated pET30/GdhB_therm and sequenced for confirmation of the gene sequence.
  • the alternative aldose dehydrogenase expressing organism was prepared by transforming BL21 (DE3) competent cells with said plasmid. The cloning procedure was performed with a target to allow expression of protein having sequence SEQ ID NO. 08.
  • SEQ ID NO. 08 which compared to SEQ ID NO 06 has altered sequence coding Ser residue at position 231 to Lys residue.
  • Procedure 1A The procedure of expression of E. coli GdhB_therm was undertaken as described in Procedure 1A. After expression cells were harvested and whole cell catalysts were obtained as described in Procedure 1B to obtain living whole cell catalysts. To obtain resting whole cell catalysts Procedure 1C was undertaken.
  • E . coli BL21(DE3) having an expression plasmid vector pET30a(+) (Novagen Inc., Madison, WI, USA) was used as negative control.
  • the control cells were prepared by transforming E. coli BL21 (DE3) competent cells with said plasmid and kanamycin resistant colonies were cultured.
  • the procedure of cultivation of E. coli BL21(DE3) pET30 was undertaken regarding to Procedure 1A. After expression cells were harvested and negative control cells were obtained as described in Procedure 1B to obtain negative control for living whole cell catalysts. To obtain negative control cells for resting whole cell catalysts Procedure 1C was undertaken.
  • E. coli Ylil, E. coli Ylil_C, E. coli GdhB or E. coli GdhB_therm (as described in Example 1, more particularly after undertaking Procedure 1A) cells were harvested by centrifugation (10 000 g, 5 min, 4°C). Supernatant was discarded after centrifugation and pellet was resuspended in phosphate buffer (50 mM KH 2 PO 4 , 150 mM NaCl, pH 7.0) in one tenth of initial volume. Cells were supplemented with 1 mg/mL lysozyme solution. Lysis was performed at 37°C, 1h. After lysis cell debris were removed by sedimentation (10 min, 20 000 g, 4 °C) to obtain a clear aqueous solution. Aldose dehydrogenase comprised within a cell free extract was thus obtained.
  • the pellet was resuspended in a lytic buffer (50 mM NaH2PO4, pH 7.0, 300 mM NaCl, 2 mM DTT) using 200 g of pellet per 1L of said buffer.
  • a lytic buffer 50 mM NaH2PO4, pH 7.0, 300 mM NaCl, 2 mM DTT
  • Cells were sonified (3 x 15 s) using Branson digital sonifier and cell debris were removed by sedimentation (10 min, 20 000 g, 4 °C) to obtain a clear aqueous solution. Aldose dehydrogenase comprised within a cell free extract was thus obtained.
  • cell free lysates were prepared from whole cell Klyuvera intermedia and Gluconobacter oxydans (cultivations and preparations of whole cell catalysts are described in Example 9 and Example 10, respectively).
  • E . coli Ylil, E . coli GdhB or E . coli GdhB_therm (as described in Example 1, more particularly after undertaking Procedure 1A) the following procedure was used. Cells from freshly expressed culture were pelleted by centrifugation and the growth medium was completely removed. The cell pellet was washed three times in 20 mM Tris-HCl (pH 7.5). The cell pellet was then centrifuged (10 000 g, 5 min, 4 °C) before resuspended in one tenth of initial culture volume of hypertonic solution containing 20 mM Tris HCl (pH 7.5), 20 % sucrose, and 0.5 mM EDTA.
  • the cell suspension was incubated on ice for 10 min. After centrifugation (10 min, 12 000 g, 4 °C) cell pellet was resuspended gently by pipetting into hypotonic solution (50 mM Tris-HCl pH 7.0) in the same volume as hypertonic solution. Cells were incubated on ice for additional 10 min. Cells were pelleted by centrifugation (10 min, 20 000 g, 4 °C) and the supernatant was removed and labelled as periplasmic fraction.
  • hypotonic solution 50 mM Tris-HCl pH 7.0
  • the aldolase gene deoC ( E . coli GeneBank #EG10221, locus tag b4381) comprised within whole cell catalyst can be obtained by a number of procedures, for example as described in WO2009/092702 . Nevertheless, we provide a nonlimiting example of preparation of aldolase enzyme (DERA) comprised within the whole cell.
  • DEA aldolase enzyme
  • the genomic DNA from E . coli DH5 ⁇ was isolated using Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to manufacturer's instructions. Isolation of genomic DNA was made using overnight culture of E . coli grown on LB medium at 37 °C. Amplification of gene deoC was performed by PCR using oligonucleotide primers CCGGCATATGACTGATCTGAAAGCAAGCAG (SEQ ID NO. 74) and CCGCTCAGCTCATTAGTAGCTGCTGGCGCTC (SEQ ID NO. 75).
  • Amplification was performed by Pfx 50 DNA polymerase (Invitrogen, Calsbad, CA, USA) as follows: an initial denaturation at 95°C for 10 min, followed by 30 cycles of 45 s at 94°C, 45 s at 60°C, and 60 s at 68°C. Final elongation was performed 420 s at 68°C. The resulting fragments were separated by agarose gel electrophoresis and purified. A 0.9-kb DNA fragment containing deoC (SEQ ID NO. 09) was separated by agarose gel electrophoresis and purified.
  • the product was ligated into plasmid pGEM T-Easy (Promega, Madison, WI, USA) in a T4 ligase reaction.
  • plasmid construct was cleaved with restriction endonucleases Nde I and Blp I, the resulting fragments were separated on agarose gel electrophoresis and 0.9 kb fragment containing deoC was purified.
  • An expression vector pET30a(+) (Novagen Inc., Madison, WI, USA) was cleaved using the same aforementioned restriction endonucleases and purified.
  • the 0.9-kb fragment containing deoC gene was assembled with the cleaved expression vector in a T4 ligase reaction. E .
  • coli JM109 cells were transformed with the obtained ligation reaction and kanamycin resistant colonies were cultured. Afterwards plasmid DNA was isolated. The resulting construct was designated pET30/DeoC and sequenced for confirmation of the gene sequence. The cloning procedure was performed with a target to allow expression of protein having sequence SEQ ID NO. 10.
  • the DERA aldolase expressing organism was prepared by transforming BL21 (DE3) competent cells with said plasmid.
  • kanamycin 25 ⁇ g/mL
  • Procedure 1A The procedure of expression of E . coli DeoC was undertaken as described in Procedure 1A, with a sole difference of IPTG inducer concentration being 0.5 mM. After expression cells were harvested and whole cell catalysts were obtained as described in Procedure 1B to obtain living whole cell catalysts. To obtain resting whole cell catalysts Procedure 1C was undertaken.
  • Example 5 Preparation of aldolase enzyme (DERA) and aldose dehydrogenase enzyme comprised within a whole cell culture of a single microorganism
  • a gene ylil having additional ribosomal binding site (RBS) was added into the construct pET30/DeoC resulting in construct bearing two different coding sequences organised in a single operon and under transcriptional control of IPTG inducible promoter.
  • the plasmid DNA from E . coli JM109 pET30/Ylil was isolated using Wizard Plus SV Minipreps DNA Purification Kit (Promega, Madison, WI, USA) according to manufacturer's instructions. Isolation of plasmid DNA was made using overnight culture of E . coli grown on LB medium at 37°C. The said isolated plasmid pET30/Ylil was used as a template in a PCR reaction to amplify sequence containing gene ylil and RBS derived from expression vector pET30a(+) upstream of the coding region.
  • PCR reaction was performed using oligonucleotide primers GCAGGCTGAGCTTAACTTTAAGAAGGAGATATACATATG (SEQ ID NO. 76) and GCGCGCTCAGCCTAATTGCGTGGGCTAACTTTAAG (SEQ ID NO. 77). Amplification of this fragment was performed by PCR using Pfu ULTRA II Fusion HS DNA 200 polymerase (Agilent, Santa Clara, CA, USA) as follows: an initial denaturation at 98°C for 3 min, followed by 30 cycles of 20 s at 98°C, 20 s at 55°C, and 90 s at 72°C. Final elongation was performed 180 s at 72°C.
  • the resulting fragments were separated by agarose gel electrophoresis and 1.2 kb fragment containing ylil and RBS site was purified.
  • the product was transferred to plasmid pGEM T-Easy (Promega, Madison, WI, USA) and designated pGEM/RBS_ylil.
  • the construct was cleaved with restriction endonuclease Blp I, then the resulting fragments were separated on agarose gel electrophoresis and 1.2 kb fragment containing ylil and RBS site was purified.
  • the construct pET30a/DeoC (construction is described in Example 4) was cleaved using the same aforementioned restriction endonuclease ( Blp I) and purified.
  • Shrimp Alkaline Phosphatase (SAP, purchased by Promega, Madison, WI, USA) was used to dephosphorylate the 5' phosphorylated ends of cleaved pET30a/DeoC according to manufacturer's instructions. Thus self-ligation of pET30a/DeoC was prevented.
  • the fragments were assembled in a T4 ligase reaction.
  • E . coli JM109 cells were transformed with the obtained ligation reaction and kanamycin resistant colonies were cultured and plasmid DNA was isolated.
  • the resulting construct was designated pET30/DeoC_RBS_Ylil and sequenced for confirmation of the gene sequences.
  • the organism expressing DERA aldolase and aldose dehydrogenase was prepared by transforming BL21 (DE3) competent cells with said plasmid.
  • E. coli Ylil and DERA comprised within a single whole cell culture was undertaken as described in Procedure 1A.
  • Procedures 1B and 1C living whole cell catalysts and resting whole cell catalysts, respectively, were prepared.
  • Gluconobacter oxydans (ATCC 621H) gene cluster pqqABCDE ( pqqA, pqqB, pqqC, pqqD, pqqE, GeneBank #CP000009, approximate location in the genome 1080978...1084164), which is involved in pyrroloquinoline quinone (PQQ) biosynthesis was expressed in E . coli.
  • the genomic DNA from Gluconobacter oxydans was isolated using Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to manufacturer's instructions. Isolation of genomic DNA was made using culture of G. oxydans grown on mannitol medium (10 g/L bacto yeast extract, 3 g/L peptone, 5 g/L mannitol, pH 7.0) 48 h at 26°C. The said isolated genome was used as template for amplification of gene cluster pqqABCDE and its own promoter. Amplification was performed by PCR using oligonucleotide primers GCGCGGTACCGCACATGTCGCGGATGTTCAGGTGTTC (SEQ ID NO.
  • plasmid pGEM T-Easy Promega, Madison, WI, USA.
  • E . coli JM109 cells were transformed with the obtained ligation reaction and ampicillin resistant colonies were cultured and plasmid DNA was isolated.
  • the resulting plasmid construct was designated pGEMpqqA-E and sequenced for confirmation of the gene sequences.
  • the cloning procedure was performed with a target to allow expression of genes under control of their native promoters and thus production of proteins PqqA, PqqB, PqqC, PqqD and PqqE derived from G . oxidans (SEQ ID NO. 12, 13, 14, 15, 16, respectively).
  • Kluyvera intermedia ATCC 33421, formerly Enterobacter intermedium
  • pqqABCDE pqq A , pqq B , pqq C , pqq D , pqq E , GenBank: AY216683.1
  • PQQ pyrroloquinoline quinone
  • the genomic DNA from Kluyvera intermedia was isolated using Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to manufacturer's instructions. Isolation of genomic DNA was made using culture of K. intermedia grown on VD medium overnight at 37°C. The said isolated genome was used as template for amplification of gene cluster pqqABCDE and its own promoter. Amplification was performed by PCR using oligonucleotide primers GCGCGGATCCACGCAGCATCGGGCCGTTCT (SEQ ID NO. 80) and GCGCGGTACCAAGCTTCGCTGCCGCAAAACA (SEQ ID NO.
  • coli JM109 cells were transformed with the obtained ligation reaction and ampicillin resistant colonies were cultured and plasmid DNA was isolated.
  • the resulting plasmid construct was designated pGEM/KI_pqqA-E and sequenced for confirmation of the gene sequences.
  • the cloning procedure was performed with a target to allow expression of genes under control of their native promoters and thus production of proteins PqqA, PqqB, PqqC, PqqD and PqqE derived from K . intermedia (SEQ ID NO. 18, 19, 20, 21, 22, respectively).
  • the suspension (inoculum size 5%, v/v) was then transferred to glucose minimal medium (100 mL, 5 g/L D-glucose, 2 g/L sodium citrate 10 g/L K2HP04, 3.5 g/L (NH4)2SO4, pH 7.0).
  • glucose minimal medium 100 mL, 5 g/L D-glucose, 2 g/L sodium citrate 10 g/L K2HP04, 3.5 g/L (NH4)2SO4, pH 7.0.
  • the culture was grown at 37°C, 250 rpm, 48 h.
  • the cell culture was supplemented with 1 mg/mL lysozyme solution. Lysis was performed at 37°C, 1 h. After lysis cell debris were removed by sedimentation (10 min, 20 000 g, 4°C) to obtain a clear aqueous solution. PQQ comprised within a cell free extract was thus obtained.
  • Example 7 Preparation of aldose dehydrogenase enzyme comprised within a whole cell culture of a single microorganism able to synthesize pyrroloquinone quinone (PQQ)
  • the expression plasmid bearing gene yli l and gene cluster pqqA -E was constructed.
  • Construct pET301Ylil (preparation is described in Example 1) was digested with restriction endonuclease Sph I.
  • Shrimp alkaline phosphatase (SAP, purchased by Promega, Madison, WI, USA) was used to dephosphorylate the 5' phosphorylated ends of cleaved pET30a/Ylil according to manufacturer's instructions.
  • SAP restriction endonuclease
  • the cleaved linker and cleaved construct were assembled in a T4 ligase reaction.
  • the introduction of the linker inserts additional restriction endonucleases recognition sites into the plasmid.
  • the construct pGEM/pqqA-E (according to Example 6) was cleaved with restriction endonucleases Bam HI and Kpn I, then the resulting fragments were separated on agarose gel electrophoresis and 3.4 kb fragment containing gene operon pqqABCDE was purified.
  • Construct pET30/Ylil with linker was cleaved using the aforementioned restriction endonucleases and purified.
  • the fragments (cleaved pET30/Ylil with linker and pqq A-E, respectively) were assembled in a T4 ligase reaction.
  • E. coli JM109 cells were transformed with the obtained ligation reaction and kanamycin resistant colonies were cultured and plasmid DNA was isolated.
  • the resulting plasmid construct was designated pET30/Ylil+pqqA-E and sequenced for confirmation of the gene sequences.
  • the organism able to express aldose dehydrogenase and meanwhile synthesize pyrroloquinone quinine was prepared by transforming BL21 (DE3) competent cells with said plasmid.
  • E . coli Ylil and comprised within a whole cell culture of a single microorganism able to produce PQQ was undertaken as described in Procedure 1A.
  • Procedures 1B and 1C living whole cell catalyst and resting whole cell catalyst, respectively, were prepared.
  • Cell free lysate was prepared following procedure described in Example 2.
  • Example 8 Preparation of aldolase enzyme (DERA) and aldose dehydrogenase enzyme comprised within a whole cell culture of a single microorganism able to synthesize pyrroloquinone quinone (PQQ)
  • DBA aldolase enzyme
  • PQQ pyrroloquinone quinone
  • Construct pET30/DeoC (preparation is described in Example 4) was digested with restriction endonuclease Sph I .
  • Shrimp alkaline phosphatase (SAP, purchased by Promega, Madison, WI, USA) was used to dephosphorylate the 5' phosphorylated ends of cleaved pET30a/DeoC according to manufacturer's instructions.
  • SAP restriction endonuclease
  • the cleaved linker and cleaved construct were assembled in a T4 ligase reaction.
  • the introduction of the linker inserts additional restriction endonucleases recognition sites.
  • pGEM/RBS_ylil (Example 5) was cleaved with restriction endonuclease Blp I, then the resulting fragments were separated on agarose gel electrophoresis and 1.2 kb fragment containing ylil and RBS site was purified.
  • the aforementioned construct pET30a/DeoC with linker was cleaved using the same restriction endonuclease ( Blp I) and purified.
  • Shrimp alkaline phosphatase SAP, purchased by Promega, Madison, WI, USA
  • SAP was used to dephosphorylate the 5' phosphorylated ends of cleaved plasmid construct pET30a/DeoC with linker according to manufacturer's instructions.
  • the fragments were assembled in a T4 ligase reaction.
  • E. coli JM109 cells were transformed with the obtained ligation reaction and kanamycin resistant colonies were cultured and plasmid DNA was isolated.
  • Intermediate construct was designed pET30/DeoC_RBS_Ylil with linker.
  • the construct pGEM/pqqA-E (according to Example 6) was cleaved with restriction endonucleases Bam HI and Kpn I, then the resulting fragments were separated on agarose gel electrophoresis and 3.4 kb fragment containing gene operon pqqABCDE was purified.
  • Construct pET30/DeoC_RBS_Ylil with linker was cleaved using the aforementioned restriction endonucleases and purified.
  • the fragments (cleaved pET30/DeoC_RBS_Ylil with linker and pqqA -E, respectively) were assembled in a T4 ligase reaction.
  • E . coli JM109 cells were transformed with the obtained ligation reaction and kanamycin resistant colonies were cultured and plasmid DNA was isolated.
  • the resulting plasmid construct was designated
  • FIG. 1 shows a physical map of inserted genes deoC. ylil. gene cluster pqqABCDE and additional double stranded sequences presenting ribosomal binding site (RBS) and linker sequence bearing additional recognition sites for restriction endonucleases into expression plasmid pET30a (Novagen Inc.. Madison. WI. USA).
  • Artificial construct was designated pET30/DeoC_RBS_Ylil+pqqA-E.
  • the organism able to synthesize pyrroloquinone quinine and meanwhile expressing DERA aldolase and aldose dehydrogenase was prepared by transforming BL21 (DE3) competent cells with said plasmid.
  • E . coli Ylil and DERA comprised within a single whole cell culture able to produce PQQ was undertaken as described in Procedure 1A, with a sole difference of IPTG inducer concentration being 0.5 mM.
  • Procedures 1B and 1C living whole cell catalysts and resting whole cell catalysts, respectively, were prepared. Cell free lysate was prepared following procedure the described in Example 2.
  • the cells were maintained at 30°C, 250 rpm for 16h.
  • Cell free lysate was prepared by following the procedure described in Example 2.
  • Gluconobacter oxydans ATCC #621H
  • the cells were maintained at 26°C, 250 rpm for 48 h.
  • Example 11 A method for determination of compound (II) oxidation/dehydrogenation activity using DCPIP as electron acceptor
  • This method is useful for determining aldose dehydrogenase activity (or activity of other dehydrogenases and/or oxidases). Accordingly the same method is used for screening and identifying enzymes and/or organisms capable of carrying out the reaction of oxidation/dehydrogenation of compound (II) resulting in compound (I).
  • Oxidation/dehydrogenation activity towards compound (II) can be measured using a living whole cell catalyst (regard to Procedure 1B), resting whole cell catalyst (regard to Procedure 1C), a lysate (preparation is described in Example 2) or a periplasmic fraction (Example 3) of any microorganism regardless of it being native or genetically modified microorganism.
  • a term "analysis material" includes all preparations of catalysts as described in previous examples. The results obtained using different preparations are given separately in provided tables below.
  • cell density of tested microorganisms was quantified as wet weight in mg per mL of sample.
  • the cells in a sample were separated from the broth by centrifugation (10 000 g, 10 min) and wet pellet was weighted.
  • centrifugation 10 000 g, 10 min
  • wet pellet was weighted.
  • lysate or periplasmic fraction was used as a testing fraction the data of wet cell weight of whole cell derived from was taken into account.
  • the reaction mixture (total volume was 200 ⁇ L) contained phosphate buffer pH 8.0, 50 mM substrate ((2S,4R)-4,6-dihydroxytetrahydro-2H-pyran-2-yl)methyl acetate), 0.2 mM DCPIP (Sigma Aldrich, Germany), 0.04 mM PMS (Sigma Aldrich, Germany). 50 ul of "analysed material” (50 ⁇ L) was added to the reaction mixture. Where needed (due to rapid completion of the reaction), the "analysed material” was diluted in phosphate buffer pH 8.0. All tested “analysed materials” were made in triplicates. Useful range in which the assay is linear was found to be between 10 and 400 mAU/sec.
  • coli BL21(DE3) pET30/GdhB (Example 1) 10 877 8 911 9 920 5 E. coli BL21(DE3) pET30/GdhB_therm (Example 1) 15 812 9 897 8 887 6 E . coli BL21(DE3) pET30/DeoC (Example 4) 9 175 15 158 8 45 7 E. c o li BL21(DE3) pET30/DeoC_RBS_Ylil (Example 5) 8 811 10 797 11 688 8 E .
  • coli BL21 (DE3) pET30/Ylil+pqqA-E (Example 6) 957 961 932 952 865 877 9 E . coli BL21(DE3) pET30/DeoC_RBS_Ylil+pqqA-E (Example 8) 889 905 970 954 943 922 10 Kluyvera intermedia (Example 9) 875 880 10 11 N.A. N.A. 11 Gluconobacter oxydans (Example 10) 640 680 11 13 N.A. N.A.
  • Example 12 Determination of properties of aldose dehydrogenase contained in E . coli in a presence of DCPIP
  • Discoloration of DCPIP was observed in these wells.
  • the discoloration rate was about two times faster when ((2S,4R)-4,6-dihydroxytetrahydro-2H-pyran-2-yl)methyl acetate was used as substrate than with control wells containing D-glucose or D-galactose as a substrate.
  • the second reaction mixture was prepared identically to the above with a sole difference: the pH value of assembled mixture being 6.0. (all compounds of reaction mixture were dissolved in phosphate buffer pH 6.0 and the initial pH value of reaction mixture was thus 6.0) All tested solutions were made in triplicates.
  • Glycerol is a bad substrate for aldose dehydrogenases tested here.
  • Example 13 Reconstitution of holo-enzyme aldose dehydrogenase with PQQ from various sources and assay with DCPIP as electron acceptor
  • Example 14 Oxidation of lactol ((2 S ,4 R )-4,6-dihydroxytetrahydro-2H-pyran-2-yl)methyl acetate with various aldose dehydrogenases comprised within living whole cell catalysts
  • the major product of the reaction had identical retention time and ion distribution as ((2S,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl)methyl acetate obtained by chemical oxidation as described in the art.
  • coli BL21 (DE3) pET30 38.20 g/L 0.00 g/L 19.17 g/L 4.26 g/L 10.12 g/L 12.01 g/L E. coli BL21 (DE3) pET30/Ylil 38.20 g/L 0.00 g/L 13.01 g/L 15.88 g/L 1.65 g/L 25.07 g/L E. coli BL21 (DE3) pET30/Ylil_C 38.20 g/L 0.00 g/L 15.12 g/L 8.74 g/L 6.98 g/L 17.65 g/L E.
  • E . coli BL21 (DE3) pET30 was used as negative control. Reaction was held at the same conditions as described above and at the end of the reaction no ((2S.4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl)methyl acetate was observed, however all of the provided ((2 S ,4 R )-4.6-dihydroxytetrahydro-2H-pyran-2-yl)methyl acetate remained untouched.
  • Example 15 Sequential reaction using DERA aldolase and aldose dehydrogenaze (Ylil) for the production ((2S.4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl) methyl acetate from acetyloxyacetaldehyde and acetaldehyde
  • Living whole cell catalyst E. coli BL21 (DE3) pET30/DeoC was prepared as described in Example 4 according to Procedure 1B. 5 mL of living whole cell catalyst was transferred to 100 mL Erlenmayer flask.
  • acetyloxyacetaldehyde and acetaldehyde were prepared. 600.5 mg acetyloxyacetaldehyde (chemical synthesis of said compound was performed in our laboratory and revealed 85 % purity) and 467.2 mg acetaldehyde (purchased by Fluka, USA) were dissolved in ice cold phosphate buffer pH 6.0 to final volume 10 mL.
  • reaction mixture At time 0' 1 mL of said stock solution of acetyloxyacetaldehyde and acetaldehyde was added into reaction mixture. Reaction was performed at 37 °C, 250 rpm for 3 hours on a rotary shaker.
  • Example 16 Simultaneous reaction using DERA aldolase and aldose dehydrogenaze (Ylil) for the production ((2S.4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl)methyl acetate from acetyloxyacetaldehyde and acetaldehyde
  • E . coli BL21 (DE3) pET30/Ylil and E . coli BL21 (DE3) pET30/DeoC were prepared as described in Procedure 1B. Both living whole cell catalysts were transferred to 100ml Erlenmayer flask in the same volume ratio (5 mL). Reaction mixture was supplemented with 1 ⁇ M PQQ (Sigma Aldrich, Germany).
  • acetyloxyacetaldehyde and acetaldehyde were prepared. 1.201 g acetyloxyacetaldehyde (chemical synthesis of said compound was performed in our laboratory and revealed 85 % purity) and 934.4 mg acetaldehyde (purchased by Fluka. USA) were dissolved in ice cold phosphate buffer pH 6.0 to final volume 10 mL.
  • Example 17 Simultaneous reaction comprising DERA aldolase and aldose dehydrogenase (Ylil) within a living whole cell catalyst of a single microorganism capably of synthesis pf PQQ for the production of ((2S,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl)methyl acetate from acetyloxyacetaldehyde and acetaldehyde
  • the below example provides a synthetic biological pathway provided within a living microorganism which is capable of producing highly enantiomericaly pure ((2S,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl)methyl acetate from simple and inexpensive molecules: acetyloxyacetaldehyde and acetaldehyde.
  • Living whole cell catalyst E. coli BI21(DE3) pET30/DeoC_RBS_Ylil+pqqA-E (preparation is described in Example 8) was prepared as described in Procedure 1A. Said whole cell catalyst was concentrated by centrifugation (5 000 g, 10 min) and pellet was resuspended in one tenth of initial volume of the same supernatant. Exceeded supernatant was discarded. 10 mL of said concentrated living whole cell catalyst was transferred to 100 ml Erlenmayer flask.
  • Example 18 Simultaneous reaction comprising DERA aldolase and living whole cell catalyst Kluyvera intermedia for the production ((2S,4R)4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl)methyl acetate from acetyloxvacetaldehyde and acetaldehyde
  • acetyloxyacetaldehyde and acetaldehyde were prepared. 1.201 g acetyloxyacetaldehyde (chemical synthesis of said compound was performed in our laboratory and revealed 85 % purity) and 934.4 mg acetaldehyde (purchased by Fluka. USA) were dissolved in ice cold phosphate buffer pH 6.0 to final volume 10 mL.
  • Example 19 Simultaneous reaction using DERA aldolase and aldose dehydrogenaze (Ylil) for the production of ((4 R ,6 S )-6-(chloromethyl)-4-hydroxytetrahydro-2H-pyran-2 one )
  • E. coli BL21 (DE3) pET30/Ylil and E. coli BL21 (DE3) pET30/DeoC (preparation is described in Examples 1 and 4, respectively) were prepared as described in Procedure 1B. 6 mL of said living whole cell catalyst E. coli BL21 (DE3) pET30/DeoC and 3 mL of E. coli BL21 (DE3) pET30/Ylil were transferred to 50 mL polystyrene conical tubes (BD Falcon, USA). Reaction mixture was supplemented with 1 ⁇ M PQQ (Sigma Aldrich, Germany).
  • reaction mixture At time 0' 1 mL of said stock solution of chloroacetaldehyde and acetaldehyde was added into reaction mixture - total reaction volume was thus 10 mL and starting concentrations of chloroacetaldehyde and acetaldehyde in reaction mixture were 100 mM and 225 mM, respectively. Reaction was performed for 2 hours at 37°C, 200 rpm of shaking in water bath.
  • Example 20 Simultaneous reaction using DERA aldolase and aldose dehydrogenaze (Ylil) for the production of ((4 R ,6 S )-6-(dimethoxymethyl)-4-hydroxytetrahydro-2 H -pyran-2-one)
  • E. coli BL21 (DE3) pET30/Ylil and E. coli BL21 (DE3) pET30/DeoC (preparation is described in Examples 1 and 4, respectively) were prepared as described in Procedure 1B. 6 mL of said living whole cell catalyst E. coli BL21 (DE3) pET30/DeoC and 3 mL of E . coli BL21 (DE3) pET30/Ylil were transferred to 50 mL polystyrene conical tubes (BD Falcon, USA). Reaction mixture was supplemented with 1 ⁇ M PQQ (Sigma Aldrich. Germany).
  • reaction mixture At time 0' 1 mL of said stock solution of dimethoxyacetaldehyde and acetaldehyde was added into reaction mixture - total reaction volume was thus 10 mL and starting concentrations of dimethoxyacetaldehyde and acetaldehyde in reaction mixture were 100 mM and 225 mM, respectively. Reaction was performed for 2 hours at 37°C, 200 rpm of shaking in water bath.
  • Example 21 Simultaneous reaction using DERA aldolase and aldose dehydrogenaze (Ylil) for the production of ((4 R ,6 S )-6-benzyloxymethyl)-4-hydroxytetrahydro-2 H -pyran 2-one)
  • E. coli BL21 (DE3) pET30/Ylil and E. coli BL21 (DE3) pET30/DeoC (preparation is described in Examples 1 and 4, respectively) were prepared as described in Procedure 1B. 6 mL of said living whole cell catalyst E. coli BL21 (DE3) pET30/DeoC and 3 mL of E . coli BL21 (DE3) pET30/Ylil were transferred to 50 mL polystyrene conical tubes (BD Falcon, USA). Reaction mixture was supplemented with 1 ⁇ M PQQ (Sigma Aldrich, Germany).
  • reaction mixture At time 0' 1 mL of said stock solution of benzyloxyacetaldehyde and acetaldehyde was added into reaction mixture - total reaction volume was thus 10 mL and starting concentrations of benzyloxyacetaldehyde and acetaldehyde in reaction mixture were 100 mM and 225 mM, respectively. Reaction was performed for 2 hours at 37°C. 200 rpm of shaking in water bath.
  • Example 22 Simultaneous reaction using DERA aldolase and aldose dehydrogenaze (Ylil) for the production of ((4 R ,6 R )-4-hydroxy-6-methyltetrahydro-2 H -pyran-2-one)
  • E. coli BL21 (DE3) pET30/Ylil and E. coli BL21 (DE3) pET30/DeoC (preparation is described in Examples 1 and 4, respectively) were prepared as described in Procedure 1B. 6 mL of said living whole cell catalyst E. coli BL21 (DE3) pET30/DeoC and 3 mL of E. coli BL21 (DE3) pET30/Ylil were transferred to 50 mL polystyrene conical tubes (BD Falcon, USA). Reaction mixture was supplemented with 1 ⁇ M PQQ (Sigma Aldrich, Germany).
  • acetaldehyde Stock solution of acetaldehyde was prepared. 445 mg of acetaldehyde (purchased by Fluka, USA) was dissolved in ice cold phosphate buffer pH 6.0 to final volume 5 mL.
  • reaction mixture At time 0' 1 mL of said stock solution of acetaldehyde was added into reaction mixture - total reaction volume was thus 10 mL and its final concentration in reaction mixture was 200 mM. Reaction was performed for 2 hours at 37°C, 200 rpm of shaking in water bath.
  • Example 23 High cell density production of living whole cell catalysts and one pot sequential production of ((2S,4R)-4-hdroxy-6-oxotetrahydro-2H-pyran-2-yl)methyl acetate from acetyloxyacetaldehyde and acetaldehyde
  • E. coli BL21(DE3) pET30/Ylil+pqqA-E and E. coli BL21 (DE3) pET30a/DeoC were prepared in "fed batch" bioprocess using laboratory bioreactors Infors ISF100 with maximal volume of 2L.
  • the reactors were stirred, aerated, temperature and pH controlled as described bellow. After consumption of initial substrates provided in the medium. ammonia and glucose are fed continuously to the process as nitrogen and carbon source, respectively.
  • composition and preparation of the media was as follows:
  • the initial medium was prepared according to a special protocol: KH2PO4, Fe(III)citrate, mineral solution, Zn(CH3COO)2 2H2O and (NH4)2HPO4 were sequentially added as solutions to about half of the final volume. After autoclaving (20 min at 121°C). sterile solutions of glucose, MgSO4 7H2O and kanamycin (25 mg/mL) were added after prior adjustment of the pH to 6.8 with 12.5 % (v/v) ammonium hydroxide solution. Sterile distilled water was added to adjust the final volume (1L) in the bioreactor. The above said solutions were sterilized separately by filtration (0.2 ⁇ m).
  • Feeding solutions were 12.5 % (v/v) ammonium hydroxide solution, the silicone antifoam compound BC SIMETHICONE ANTIFOAM C100EP (Basildon Chemical. UK) and 50 % (w/v) glucose.
  • VD medium 50 mL; 10 g/L Bacto yeast extract, 5 g/L glycerol, 5 g/L NaCl, 4 g/L NaH2PO4*2H2PO. pH was adjusted with 1 M NaOH to 7.0) was inoculated with a single colony of said whole cell catalyst from a freshly streaked VD agar plate and pre-cultured to late exp. phase (37 °C, 250 rpm. 8h).
  • the initial process parameters at inoculation were as follows: 25°C, air flow rate 1.5 Umin (1.5 WM), stirrer speed 800 rpm, pH 6.8.
  • the dissolved oxygen concentration was kept at ⁇ 20 % of saturation by a pO2/agitation rate control loop and a pO2/air flow ratio control loop. towards the end of bioprocess approaching to 0 % and bioreactor capabilities reaching maximum (stirrer speed 2000 rpm, aeration 3L/min).
  • the pH was kept at 6.8 during the whole process using a pH sensor controlled external pump which provides pulses of Ammonia solution to the bioreactor whenever the pH drops below 6.8.
  • Induction for expression of protein Ylil in the culture of E . coli BL21 (DE3) pET30/Ylil+pqqA-E. was performed by adding 0.05 mM IPTG (Sigma Aldrich, Germany) 6 hours after start of the feeding phase.
  • the overall length of the process is 34 - 42h and wet weight of biomass at level between 200 and 240g/L is obtained.
  • the high density culture of E . coli BL21(DE3) pET30a/DeoC was kept in the bioreactor and strirred with 800 rpm. Temperature was raised to 37°C and 56.6 g of acetyloxyacetaldehyde and 120 mL of acetaldehyde (45.4 g) diluted in water were added with programmable pump to the reaction mixture. The whole quantity of acetyloxyacetaldehyde was added in with the constant flow rate in 30 minutes.
  • Acetaldehyde was added continuously in 3 hours time span as described in the table below: Time [min] 0 5 10 15 20 25 30 35 45 60 75 90 105 120 135 150 165 180 Volume of added acetaldehyde [mL] 0 15 27 38 47 54 60 65 73 82 90 96 102 106 110 114 117 120 Flow [mL/min] 3000 2400 2 200 1 800 1 400 1 200 1 000 0 800 0600 0533 0400 0400 0267 0 267 0 267 0 200 0 200 0

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WO2013068917A1 (fr) * 2011-11-07 2013-05-16 Dr. Reddy's Laboratories Limited Procédés de production d'un intermédiaire de statine lactone par oxydation enzymatique
CN108410831A (zh) * 2018-03-22 2018-08-17 浙江工业大学 酮酸还原酶、基因、工程菌及在合成手性芳香2-羟酸中的应用

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